In vitro development of a muscle-tendon junction construct using decellularised extracellular matrix: Effect of cyclic tensile loading.

The muscle tendon junction (MTJ) plays a crucial role in transmitting the force generated by muscles to the tendon and then to the bone. Injuries such as tears and strains frequently happen at the MTJ, where the regenerative process is limited due to poor vascularization and the complex structure of the tissue. Current solutions for a complete tear at the MTJ have not been successful and therefore, the development of a tissue-engineered MTJ may provide a more effective treatment. In this study, decellularised extracellular matrix (DECM) derived from sheep MTJ was used to provide a scaffold for the MTJ with the relevant mechanical properties and differentiation cues such as the relase of growth factors. Human mesenchymal stem cells (MSCs) were seeded on DECM and 10 % cyclic strain was applied using a bioreactor. MSCs cultured on DECM showed significantly higher gene and protein expression of MTJ markers such as collagen 22, paxillin and talin, than MSCs in 2D culture. Although collagen 22 protein expression was higher in the cells with strain than without strain, reduced gene expression of other MTJ markers was observed when the strain was applied. DECM combined with 10 % strain enhanced myogenic differentiation, while tenogenic differentiation was reduced when compared to static cultures of MSCs on DECM. For the first time, these results showed that DECM derived from the MTJ can induce MTJ marker gene and protein expression by MSCs, however, the effect of strain on the MTJ development in DECM culture needs further investigation.

Full-Field Strain Measurements of the Muscle-Tendon Junction Using X-ray Computed Tomography and Digital Volume Correlation.

We report, for the first time, the full-field 3D strain distribution of the muscle-tendon junction (MTJ). Understanding the strain distribution at the junction is crucial for the treatment of injuries and to predict tear formation at this location. Three-dimensional full-field strain distribution of mouse MTJ was measured using X-ray computer tomography (XCT) combined with digital volume correlation (DVC) with the aim of understanding the mechanical behavior of the junction under tensile loading. The interface between the Achilles tendon and the gastrocnemius muscle was harvested from adult mice and stained using 1% phosphotungstic acid in 70% ethanol. In situ XCT combined with DVC was used to image and compute strain distribution at the MTJ under a tensile load (2.4 N). High strain measuring 120,000 µÎµ, 160,000 µÎµ, and 120,000 µÎµ for the first principal stain (εp1), shear strain (γ), and von Mises strain (εVM), respectively, was measured at the MTJ and these values reduced into the body of the muscle or into the tendon. Strain is concentrated at the MTJ, which is at risk of being damaged in activities associated with excessive physical activity.

Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation.

Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization.