Understanding the structure and mechanics of the sheep calcaneal enthesis: a relevant animal model to design scaffolds for tissue engineering applications

Tendon or enthesis injuries are a worldwide clinical problem. Along the enthesis, collagen fibrils show a progressive loss of anisotropy and an increase in mineralization reaching the bone. This causes gradients of mechanical properties. The design of scaffolds to regenerate these load-bearing tissues requires validation in vivo in relevant large animal models. The sheep tendon of triceps surae muscle is an optimal animal model for this scope with limited knowledge about its structure and mechanics. We decided to investigate in-depth its structure and full-field mechanics. Collagen fibrils morphology was investigated via scanning electron microscopy revealing a marked change in orientation/dimensions passing from the tendon to the enthesis. Backscatter electron images and nanoindentation at the enthesis/bone marked small gradients of mineralization at the mineralized fibrocartilage reaching 27%wt and indentation modulus around 17-30 GPa. The trabecular bone instead had indentation modulus around 15-22 GPa. Mechanical tensile tests with digital image correlation confirmed the typical non-linear behavior of tendons (failure strain = 8.2 ± 1.0 %; failure force = 1369 ± 187 N) with maximum principal strains reaching mean values of ε_p1 ∼ 7 %. The typical auxetic behavior of tendon was highlighted by the minimum principal strains (ε_p2 ∼ 5 %), progressively dampened at the enthesis. Histology revealed that this behavior was caused by a local thickening of the epitenon. Cyclic tests showed a force loss of 21 ± 7 % at the last cycle. These findings will be fundamental for biofabrication and clinicians interested in designing the new generation of scaffolds for enthesis regeneration.