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

Tendon/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 of being validated 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 understand 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 tendon to 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. Graphical Abstract Statement of Significance Tendon and enthesis lesions are a clinical problem. To validate scaffolds for these applications large animal models are needed. Sheep tendon of triceps surae muscle is an optimal site for this scope. However, little is known about its extracellular matrix structure and mechanical properties. This work investigates the structure and mechanics of this tissue from different points of view. Scanning electron microscopy and histology studied its extracellular matrix morphology and composition. Backscattered electron images and nanoindentation assessed gradients of mineralization and stiffness at the enthesis. Mechanical tensile and cyclic tests coupled with digital image correlation elucidated its mechanics and superficial strain distribution. These findings will be fundamental for biofabrication and clinician experts to design innovative scaffolds to regenerate the enthesis.