Multiscale mechanisms of tendon fatigue damage progression and severity are strain and cycle dependent.

Tendinopathies are common chronic injuries that occur when damage accumulation caused by sub-rupture fatigue loading outpaces repair. Studies have linked fatigue loading with various mechanical, structural, and biological changes associated with pathology. However, the multiscale progression of damage accumulation with respect to area, severity and the distinct contributions of strain level and number of cycles has not been fully elucidated. The objective of this study was to investigate multiscale mechanisms underlying fatigue damage accumulation and their effect on the cellular environment. Using an in situ model in rat tail tendon (RTT), fatigue loading was applied at various strains and cycle numbers to induce fatigue damage. Pre- and post- fatigue diagnostic mechanical testing, second harmonic generation (SHG) imaging, and transmission electron microscope (TEM) imaging were used to investigate extracellular and cellular damage modes at multiple scales. Fatigue loading at strains at or below 1.0% resulted in no significant changes in SHG damage area or severity and no changes in collagen fibril or cell morphology compared with controls. Fatigue loading at strains above 1.5% resulted in greater mechanical changes correlated with increased damage area measured by SHG and collagenous damage observed by TEM. Increased cycles at high strain further altered mechanical properties, increased structural damage severity (but not area), and altered TEM collagen rupture patterns. Cell morphology was similarly progressively affected with increased strain and cycle number. These damage mechanisms that may trigger degenerative changes characteristic of tendinopathy could be targeted as a part of prevention or therapy.

Tendon extracellular matrix damage detection and quantification using automated edge detection analysis.

The accumulation of sub-rupture tendon fatigue damage in the extracellular matrix, particularly of type I collagen fibrils, is thought to contribute to the development of tendinopathy, a chronic and degenerative pathology of tendons. Quantitative assessment of collagen fibril alignment is paramount to understanding the importance of matrix injury to cellular function and remodeling capabilities. This study presents a novel application of edge detection analysis to calculate local collagen fibril orientation in tendon. This technique incorporates damage segmentation and stratification by severity which will allow future analysis of the direct effect of matrix damage severity on the cellular and molecular response.

Relating applied strain to the type and severity of structural damage in the rat median nerve using second harmonic generation microscopy.

INTRODUCTION: Stretch injuries in peripheral nerves can cause pain, paralysis, and loss of sensation. Although optimal treatment depends on the degree of injury, it is difficult to determine the severity of induced nerve damage. METHODS: The load-deformation curves of rat median nerves were generated from monotonic load-to-failure experiments to determine low, medium, and high strain levels. Additional excised median nerves were then elongated to induce damage at low (4%), medium (10% and 12%), and high (14% and 20%) tensile strains and the resulting structural damage was evaluated using second harmonic generation (SHG) imaging and light microscopy. RESULTS: No substantial structural changes occurred at 4% strain, but higher strain values resulted in disruption of the normal collagen architecture. CONCLUSIONS: The results demonstrate a spectrum of structural damage that can be monitored using SHG, a non-destructive imaging modality, and that the pattern of damage may correspond to functional deficit.

Cycle-dependent matrix remodeling gene expression response in fatigue-loaded rat patellar tendons.

Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low-cycle (100) and high-cycle (7,200) sub-failure-fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real-time RT-PCR at 1- and 7-day post-loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high-cycle group showed upregulation of TIMP-1, -2, Col3a1, and Col5a1, and downregulation TIMP-4 at both time points, upregulation of MMP-2 at 7-day post-loading and downregulation of MMP-13 and -14 at 1-day post-loading, suggesting overall repair/remodeling. In contrast, the low-cycle loaded group showed upregulation of MMP-2, -3, -13, and Col12a1 at both time points, upregulation of TIMP-1, -2, -3, Col3a1, and integrin ß1 and downregulation of integrin α11 at 1-day post-loading and upregulation of Col1a1 at 7-day post-loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high- and low-cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response.

Automated image analysis method for quantifying damage accumulation in tendon.

Tendon pathology is frequently sub-clinical prior to frank rupture, denoting the need for non-destructive methods of assessing disease presence and progression. Despite the lack of clinical presentation, previous studies have observed that distinct changes to the tendon microstructure are present, and that such qualitative changes have a dose-response relationship with the level of damage accumulated. These initial findings suggest that there is value in investigating the physical nature of damage within tendon, not only to better understand the pathological process, but also to gain insight into reparative processes and develop treatments. However, a necessary first step towards carrying out these avenues of research is to develop diagnostic tools for quantitatively assessing the level of damage present. In this study, we established a dose-response relationship between a quantitative measure of structural damage and the level of global damage induced. Furthermore, we developed and validated an automated technique for quantifying matrix disorganization (damage), which correlates and co-localizes strongly with manual quantification. In combination, these findings allow us to measure the amount of damage accumulation of a region of tendon on a clinical scale, rapidly and accurately.

Tendon-derived stem/progenitor cell aging: defective self-renewal and altered fate.

Aging is a major risk factor for tendon injury and impaired tendon healing, but the basis for these relationships remains poorly understood. Here we show that rat tendon- derived stem / progenitor cells (TSPCs) differ in both self-renewal and differentiation capability with age. The frequency of TSPCs in tendon tissues of aged animals is markedly reduced based on colony formation assays. Proliferation rate is decreased, cell cycle progression is delayed and cell fate patterns are also altered in aged TSPCs. In particular, expression of tendon lineage marker genes is reduced while adipocytic differentiation increased. Cited2, a multi-stimuli responsive transactivator involved in cell growth and senescence, is also downregulated in aged TSPCs while CD44, a matrix assembling and organizing protein implicated in tendon healing, is upregulated, suggesting that these genes participate in the control of TSPC function.