Quantifying the role of hyaluronic acid on collagen fiber mechanics via femtosecond laser ablation

Despite therapeutic use of hyaluronic acid (HA) for tendon injuries and tendinopathies, the role of HA in tendon mechanical behavior remains poorly understood. Here, we describe the development of a method that integrates femtosecond laser ablation, second harmonic generation imaging, and mechanical testing to quantify collagen fiber recoil within intact murine tendons under load. Fiber recoil following ablation is used as a measure of collagen fiber sliding and local viscoelastic behavior. Aging and enzymatic depletion of endogenous HA both significantly reduce tendon HA content and decrease elastic, viscoelastic, and total collagen fiber recoil, indicating diminished fiber sliding. To restore HA, low molecular weight methacrylated HA (MeHA) is diffused into tendons and retained through photocrosslinking. MeHA supplementation recovers collagen fiber recoil in HA-depleted and aged tendons, restoring micromechanical behavior toward adult control levels. Quasi-linear viscoelastic modeling further reveals HA-dependent changes in relaxation behavior that are not evident from conventional macroscale mechanical measurements alone. These findings identify femtosecond laser ablation as a powerful platform to quantify fiber-scale mechanics and investigate extracellular matrix contributions to tissue material properties, the results of which specifically isolate HA as an important contributor to tendon collagen fiber sliding and mechanical properties.