Structure-function specialisation of the interfascicular matrix in the human achilles tendon

Tendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency, but such tendons commonly obtain debilitating injuries. In equine tendons, energy storing is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the human positional anterior tibial tendon and energy storing Achilles tendons, testing the hypothesis that the Achilles tendon IFM has specialised composition and mechanical properties, which are lost with ageing. Data demonstrate IFM specialisation in the energy storing Achilles, with greater elasticity and fatigue resistance than in the positional anterior tibial tendon. With ageing, alterations occur predominantly to the proteome of the Achilles IFM, which are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments for tendinopathy

Structure and collagen crimp patterns of functionally distinct equine tendons, revealed by quantitative polarised light microscopy (qPLM)

Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons. IFM shows lower birefringence (0.0013 ± 0.0001 [-]), as compared to fascicles (0.0044 ± 0.0005 [-]), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components. By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity

The nanoscale architecture of force-bearing focal adhesions

The combination of micropillar array technology to measure cellular traction forces with super-resolution imaging allowed us to obtain cellular traction force maps and simultaneously zoom in on individual focal adhesions with single-molecule accuracy. We achieved a force detection precision of 500 pN simultaneously with a mean single-molecule localization precision of 30 nm. Key to the achievement was a two-step etching process that provided an integrated spacer next to the micropillar array that permitted stable and reproducible observation of cells on micropillars within the short working distance of a high-magnification, high numerical aperture objective. In turn, we used the technology to characterize the super-resolved structure of focal adhesions during force exertion. Live-cell imaging on MCF-7 cells demonstrated the applicability of the inverted configuration of the micropillar arrays to dynamics measurements. Forces emanated from a molecular base that was localized on top of the micropillars. What appeared as a single adhesion in conventional microscopy were in fact multiple elongated adhesions emanating from only a small fraction of the adhesion on the micropillar surface. Focal adhesions were elongated in the direction of local cellular force exertion with structural features of 100–280 nm in 3T3 Fibroblasts and MCF-7 cells. The combined measure of nanoscale architecture and force exerted shows a high level of stress accumulation at a single site of adhesion

Mice with a heterozygous Lrp6 deletion have impaired fracture healing

Bone fracture non-unions, the failure of a fracture to heal, occur in 10%–20% of fractures and are a costly and debilitating clinical problem. The Wnt/β-catenin pathway is critical in bone development and fracture healing. Polymorphisms of linking low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt-binding receptor, have been associated with decreased bone mineral density and fragility fractures, although this remains controversial. Mice with a homozygous deletion of Lrp6 have severe skeletal abnormalities and are not viable, whereas mice with a heterozygous deletion have a combinatory effect with Lrp5 to decrease bone mineral density. As fracture healing closely models embryonic skeletal development, we investigated the process of fracture healing in mice heterozygous for Lrp6 (Lrp6 (+/−)) and hypothesized that the heterozygous deletion of Lrp6 would impair fracture healing. Mid-diaphyseal femur fractures were induced in Lrp6 (+/−) mice and wild-type controls (Lrp6 (+/+)). Fractures were analyzed using micro-computed tomography (μCT) scans, biomechanical testing, and histological analysis. Lrp6 (+/−) mice had significantly decreased stiffness and strength at 28 days post fracture (PF) and significantly decreased BV/TV, total density, immature bone density, and mature area within the callus on day-14 and -21 PF; they had significantly increased empty callus area at days 14 and 21 PF. Our results demonstrate that the heterozygous deletion of Lrp6 impairs fracture healing, which suggests that Lrp6 has a role in fracture healing