Fig. 2. Multiscale toughening mechanisms enable the entheses to exhibit distinct failure modes under varying loading conditions.

(A and B), To examine the effect of loading on failure mode, samples were loaded (A) across a range of loading rates to simulate acute injuries or (B) loaded cyclically to simulate degenerative loading. (C) Enthesis strength (i.e., failure load) and (D) enthesis toughness (i.e., energy absorption) increased with the loading rate. *P< 0.05, analysis of variance (ANOVA) followed by the Dunnet’s multiple comparison test]. (E) There were three distinct failure modes, depending on the loading regime: bone avulsion, tendon mid-substance, and tendon-bone interface (insertion failure). Scale bars, 500 μm. Under monotonic loading, most samples failed by bony avulsion failures. Under “high” cyclical loading (20 to 70% failure force), all samples failed at the insertion. Under “low” cyclical loading (1 to 20% failure force), samples did not fail, even after 100,000 cycles. (F) F-CHP fluorescence intensity, indicative of collagen damage accumulation, increased with the level of applied load and with the number of cycles. For quasi-statically loaded samples (top), there was little to no fluorescent signal in the low force group (1 to 2 N), followed by increased staining near the attachment site at higher loads (3 N and failure). For cyclically loaded samples (bottom), F-CHP staining was initially concentrated in a few fibers near the tendon mid-substance (10 to 40 K cycles) and ultimately propagated down the entire tendon in concentrated bands. Scale bars, 500 μm.