Szczesny, S. E. | Search bioRxiv

bioRxiv2026

Biological Signaling Pathways Associated with Late Tendon Development in Chicken Embryos and Their Dependence on Mechanical Stimulation

During embryonic and neonatal development, tendons exhibit rapid changes in mechanics and structure, including increases in collagen content, crosslinking, and fibril length. These functional and structural changes are partly dependent on mechanical stimulation. However, the specific mechanobiological mechanisms driving these changes remain poorly understood. The objective of this study was to identify the potential biological mechanisms and pathways driving tendon maturation into load-bearing materials using an embryonic chick model. Using bulk RNA sequencing, gene set enrichment analysis, and transcription factor analysis, we produced a transcriptomic profile of chick hindlimb tendons during late embryonic development (E16, E18, and E20) and tendons from rigid and flaccid paralysis-treated embryos. Additionally, we assessed the results from two differential expression analysis models to improve the replicability of our findings. Our results suggest that the largest transcriptomic response occurs between embryonic day 16 and 20, and that among paralysis conditions, rigid paralysis produces the greatest effect relative to controls. Across development, we found positive enrichment in processes related to mechanotransduction, extracellular matrix remodeling, cell adhesion, and contractility, and identified candidate pathways upregulated with development, including TGF-{beta}, PI3K-AKT-mTOR, Hippo, circadian rhythm, and focal adhesion signaling. In paralysis-treated embryos, our data suggest impairment in extracellular matrix remodeling and mechanotransduction events. Specifically, we found that both modes of paralysis are associated with a cellular stress response and downregulation of WNT signaling. Together, our findings identify candidate mechanotransduction pathways that are associated with late tendon maturation and altered by paralysis, which may inform tendon tissue engineering and regenerative approaches.

bioengineering↗

bioRxiv2021

Mechanical Stimulation via Muscle Activity is Necessary for the Maturation of Tendon Multiscale Mechanics during Embryonic Development

During embryonic development, tendons transform into a hypocellular tissue with robust tensile load-bearing capabilities. Previous work suggests that this mechanical transformation is due to increases in collagen fibril length and is dependent on mechanical stimulation via muscle activity. However, the relationship between changes in the microscale tissue structure and changes in macroscale tendon mechanics is still unclear. Additionally, the specific effect of mechanical stimulation on the multiscale structure-function relationships of developing tendons is also unknown. Therefore, the objective of this study was to measure the changes in tendon mechanics and structure at multiple length scales during embryonic development with and without skeletal muscle paralysis. Tensile testing of tendons from chicken embryos was performed to determine the macroscale tensile modulus as well as the magnitude of the fibril strains and interfibrillar sliding with applied tissue strain. Embryos were also treated with either decamethonium bromide or pancuronium bromide to produce rigid or flaccid paralysis. Histology was performed to assess changes in tendon size, spacing between tendon subunits, and collagen fiber diameter. We found that the increase in the macroscale modulus observed with development is accompanied by an increase in the fibril:tissue strain ratio, which is consistent with an increase in collagen fibril length. Additionally, we found that flaccid paralysis reduced the macroscale tendon modulus and the fibril:tissue strain ratio, whereas less pronounced effects that were not statistically significant were observed with rigid paralysis. Finally, skeletal paralysis also reduced the size of collagen fibril bundles (i.e., fibers). Together, these data suggest that more of the applied tissue strain is transmitted to the collagen fibrils at later embryonic ages, which leads in an increase the tendon macroscale tensile mechanics. Furthermore, our data suggest that mechanical stimulation during development is necessary to induce structural and mechanical changes at multiple physical length scales. This information provides valuable insight into the multiscale structure-function relationships of developing tendons and the importance of mechanical stimulation in producing a robust tensile load-bearing soft tissue.

developmental biology↗