Anterior cruciate ligament overuse increases collagen fibril compliance: collagen unraveling and elevated glycosaminoglycan activity in a murine model

Abstract

Data increasingly indicates that many anterior cruciate ligament (ACL) injuries are likely due to tissue damage accumulation and the gradual decrease in ACL strength and stiffness over time. Previous ex vivo experiments found molecular-level ACL damage (collagen unraveling) to be associated with increased tissue compliance at the collagen fibril-level. However, it remains unclear if this biomechanical effect is present in vivo, or what effect other modulators of tissue mechanics (e.g., glycosaminoglycans (GAGs)) have in fatigued-damaged ACL matrices. Herein, we investigated the in vivo relationship between collagen unraveling and fibril-level mechanics in response to ACL fatigue loading in a mouse model and determined whether GAGs are also involved in altering tissue mechanics in this injury scenario. Mouse ACLs were submaximally fatigue loaded in vivo and harvested after one hour of tissue recovery. Compared to their contralateral controls, fatigued ACLs showed increased collagen unraveling and elevated GAG signatures within damaged extracellular matrix (ECM) regions. These regions also exhibited higher fibril-level compliance. Spectral data indicated colocalization of unraveled collagen and GAG-rich domains. Versican, a large aggregating PG, emerged as a candidate contributor to increased GAG presence in these regions. Together, these findings suggest that fatigue-induced collagen unraveling promotes local GAG accumulation – mediated by PGs such as versican – which likely contributes to reinforcement of the ECM.

Statement of significance

Outcomes from this anterior cruciate ligament (ACL) fatigue loading study provide high-resolution in vivo evidence for a structural and biochemical mechanism for the development of chronic overuse injuries. Our findings suggest that fatigue-induced tropocollagen unraveling increases collagen fibril-level tissue compliance, and that glycosaminoglycans rapidly colocalize with these damaged regions, further influencing ACL mechanics. The large aggregating proteoglycan, versican, was immunohistochemically identified as a candidate for glycosaminoglycan recruitment to the damaged extracellular matrix. Versican may be preferentially retained in domains of tropocollagen damage to facilitate the tissue remodeling response, though further research is needed to confirm this potential link.