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The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0–1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell–cell communication after overstretching. [ABSTRACT FROM AUTHOR]

Abstract: Cyclic and static loading regimes are commonly used to study tenocyte metabolism in vitro and to improve our understanding of exercise-associated tendon pathologies. The aims of our study were to investigate if cyclic and static stress relaxation affected the mechanical properties of tendon fascicles differently, if this effect was reversible after a recovery period, and if the removal of glycosaminoglycans (GAGs) affected sample recovery. Tendon fascicles were dissected frombovine-foot extensors and subjected to 14% cyclic (1Hz) or static tensile strain for 30min. Additional fascicles were incubated overnight in buffer with 0.5U chondroitinase ABC or in buffer alone prior to the static stress-relaxation regime. To assess the effect of different stress-relaxation regimes, a quasi-static test to failure was carried out, either directly post loading or after a 2h recovery period, and compared with unloaded control fascicles. Both stress-relaxation regimes led to a significant reduction in fascicle failure stress and strain, but this was more pronounced in the cyclically loaded specimens. Removal of GAGs led to more stress relaxation and greater reductions in failure stress after static loading compared to controls. The reduction in mechanical properties was partially reversible in all samples, given a recovery period of 2h. This has implications for mechanical testing protocols, as a time delay between fatiguing specimens and characterization of mechanical properties will affect the results. GAGs appear to protect tendon fascicles from fatigue effects, possibly by enabling sample hydration. [Copyright &y& Elsevier]

Abstract: Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1Hz for 48h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy. [Copyright &y& Elsevier]