Achilles tendon mechanical properties depend on a complex hierarchical design, with collagen being the smallest load-bearing unit. At the nanoscale, collagen molecules are organized into fibrils, which at the microscale are assembled into fibers, followed by larger structures such as sub-tendons or fascicles. Degree of in vivo loading affects the collagen content, and organization and consequently the tissue's mechanical response. We aim to unravel how composition, structural organization, and mechanical response are affected by degree of in vivo loading at each length scale. The presentation will outline the results to date about to the use of high-resolution synchrotron-based tissue characterisation methods on several length scales in combination with in situ mechanical tests. We use a rat model, where the tendons are subjected to varying loading in vivo. To characterize the tissue microstructure, phase-contrast enhanced synchrotron micro-tomography is performed. The 3D fiber organization in fully loaded tendons is highly aligned, whereas the fibers in unloaded tendons are significantly more heterogeneously arranged and crimped. To characterize the collagen fibril response, Small Angle X-ray Scattering is performed. Two types of fibril organizations are found; a single population oriented towards the main load direction and two fibril subpopulations with clearly distinct orientations. Scattering during loading showed that the fibrils in unloaded tendons did not strain as much in fully loaded. In situ loading concurrently with high resolution synchrotron experiments show the complex tendon response to in situ load and its relation to in vivo loading and tendon hierarchical structure. Unloading seems to alter the organization of the fibrils and fibers, e.g. increased crimping and more pronounced sub-tendon twists.

Acknowledgements: Funding from Knut and Alice Wallenberg Foundation and European Research Council (101002516). Paul Scherrer Institut, Switzerland for beamtime at cSAXS and TOMCAT.

Introduction

The healing of Achilles tendon rupture is slow and jogging is usually allowed already 6 months after injury. However, the metabolic status of the healing tendon is largely unknown at the time-points when increased loading is allowed. The purpose of this study was to investigate tendon metabolic response and blood flow at 3, 6 and 12 months after Achilles tendon rupture by positron emission tomography (PET) and ultrasound-Power Doppler (UPD).

Healing of tendons is sensitive to mechanical loading, and the callus strength is reduced by ¾ after 14 days, if loading is prevented. Exogenous GDFs stimulate tendon healing. This response is influenced by loading: without loading, cartilage and bone formation is initiated. This suggests that BMP signalling is crucial during tendon healing, and that it is influenced by mechanical loading. We investigated if mechanical loading influences BMP signalling in intact and healing tendons, and how BMP gene expression changes during healing.

The Achilles tendon was transected in rats and left to heal. Half of the rats had one Achilles tendon unloaded by injection of Botox in the calf muscles. Ten tendons were analyzed before transection and for each of four time points. Gene expression for OP-1, GDF-5, -6, -7, Follistatin, Noggin, BMP-receptor 1b and BMP-receptor 2 were analysed with real-time PCR.

Loading had no detectable effects on intact tendons. During repair, loading decreased follistatin by more than half (p=0.0001), and increased GDF-5 (p=0.02). All genes showed changes during repair (p=0.0001), but the time sequences differed. GDF-5 and GDF-7 were generally more expressed than OP-1 and GDF-6. GDF-5 and GDF-7 were more expressed in normal tendons than during repair. Noggin was never detected.

Our results suggest that GDF-5 is specific for the mature tendon, and not much involved in repair. This contrasts to GDF-7, which is involved in both. OP-1 and GDF-6 seem to be involved in early healing. There was less expression of follistatin in loaded tendons during healing. The mechanosensitivity is likely of most importance at day 14 and 21 since the difference in strength between loaded and unloaded tendons is huge. An Anova with only these time points reveals effects of loading on GDF-5 and follistatin (p=0.0001 for both) and significant differences between the days for most variables.