| Tendons are a principal tissue involved in movement and function, primarily to transfer force from muscle to bone. Tendon injuries markedly limit biomechanical function, and current treatments rely on grafting which have significant drawbacks. Tissue engineering strategies remain an attractive alternative, but have not yet produced a functional tendon. We postulate that this has been due to a limited understanding of tendon development and its mechanical micro-environment. We hypothesize that tendon tissue elastic modulus changes during embryonic development, that collagen crosslinking mediates these mechanical changes, and that tendon elastic modulus is a regulator of tendon cell differentiation.;To test this hypothesis, we characterized the mechanical and biochemical development of embryonic chick tendon, and evaluated the potential of tissue engineering scaffolds with embryonic tendon modulus to regulate embryonic tendon cell behavior. We first quantified the elastic modulus of embryonic chick tendon using atomic force microscopy, and found that modulus increases non-linearly during development. Temporally correlating these changes to DNA, glycosaminoglycan and collagen content suggested that collagen content may be a contributor to tendon mechanical development, though the correlation was weak. A spatial correlation analysis supported these findings. Probing further, we inhibited lysyl oxidase (LOX) activity to prevent collagen crosslinking, which produced significant reductions in tendon elastic modulus. LOX inhibition also produced marked reductions in crosslinking density, as evaluated by mass spectrometry and multiphoton microscopy. These results indicated that collagen crosslinking contributes significantly to the development of tendon mechanical properties. In particular, we found that the hydroxylysyl pyridinoline-to-dry mass ratio is a robust functional marker of tendon development. Finally, we found that elastic modulus regulates embryonic tendon cell actin morphology and tenogenic gene expression in engineered 3-dimensional hydrogels, suggesting that physical properties may regulate tenogenesis. Taken together, this work enables the design of scaffolds that mimic the mechanical and biochemical properties of developing tendon, which may encourage stem cell tenogenesis and new matrix synthesis in vitro. In addition, these properties may serve as functional markers to assess whether engineered tendon formation proceeds in a manner similar to normal tendon development, acting as a guide for tendon regeneration strategies. |
