gms | German Medical Science

Objectives: High stress peaks occur at interfaces between soft and hard materials resulting in the interfaces' susceptibility to rupture. Creating durable soft-hard interfaces is not only a challenge in medicine, where surgical fixation of soft tissue to bone is often followed by retear of the tissue interface, but also in other disciplines, such as engineering. Despite the failure risk of most soft-hard interfaces, the Achilles tendon enthesis, the junction between Achilles tendon and calcaneus, shows outstanding strength and resilience despite undergoing high loads and angle changes. Biomimicry of the Achilles tendon enthesis' unique features could lead to new approaches for interface tissue engineering as well as for combining hard and soft materials in structural engineering. In this combined structural and compositional study we thoroughly characterize a sophisticated biomaterial to understand how soft and hard materials can be efficiently joined.

Methods: We performed a combined characterization of structure, molecular composition, and function of the porcine Achilles tendon enthesis. Firstly, we analyzed distribution of collagen type I and II via confocal imaging of immunofluorescently labeled cryosections. Secondly, we investigated the structural arrangement of fibers using scanning electron microscopy, confocal imaging and nano computed tomography (nanoCT). Thirdly, we show by modeling how the structures observed reduce local stress concentrations at the interface region. Fourthly, we characterize the full protein composition of the bone-tendon interface region using tandem mass spectrometry (LC-MS/MS).

Results and Conclusion: Confocal imaging, electron microscopy and nanoCT unambiguously show that tendon fibers spread into subfibers at the interface region ~500µm before attaching to bone. Using confocal reflectance microscopy, we show that there is a complimentary localization of collagen type I and II at the interface and that the collagen type II-rich region coincides with the region of fiber spreading. Fibers undergo a structural alteration in fiber geometry and a compositional change from collagen type I to collagen type II before inserting into bone. By modeling, we show that the observed change in fiber geometry reduces local stresses at the interface region thereby attenuating the enthesis' susceptibility to rupture. Furthermore, we identify >400 proteins at the Achilles tendon bone interface. 18 proteins show statistically significant (p<0.05) higher levels at the interface region than in the tendon, 9 proteins are statistically significant (p<0.05) more abundant in the tendon.

This study on the interplay of architecture, biochemical composition, and function of the Achilles tendon enthesis provides a guideline for biomimetic strategies to design hard-soft interfaces. The thorough characterization may not only help in designing hard-soft tissue engineering scaffolds, but also aid in efficiently joining hard and soft materials in engineering.