Structural characterization of newt tendon regeneration after complete transection: In vivo two‐photon imaging and transmission electron microscopy

Mammalian tendons, including human tendons, possess limited regeneration capability, and its healing results in scar tissue formation. However, it was recently shown that tendons of newts, amphibians that exhibit regeneration capacity in various tissues and organs, achieve full regeneration, structurally and functionally, following complete transection. The present study was performed to characterize newt tendon regeneration structurally at both micro- and nanoscales following transection surgery. In particular, we observed the progress of tendon regeneration in the same newt by developing a unique, live, in vivo imaging technique. Initial cell infiltration and formation of granulation-like tissue were evident between residual tendon stubs during the first week of regeneration. This newly formed tissue bridged tendon stubs by 3 weeks. This was followed by remodeling of the initial matrix to new tendon from the 6th to 12th week, during which the mechanical properties of regenerated tendon reached levels equivalent to those of normal tendons. These microscopic structural changes were associated with ultrastructural maturation. Collagen fibril density and fibril area fraction at the nanoscale were significantly improved from the 3rd to 6th week, and fibril area fraction at the microscale was significantly improved from 6 to 12 weeks. Such changes were not observed in a mouse model. These experimental findings suggest that newt tendon regeneration can be divided into two phases: the early phase (<6 weeks) and the late phase (≥6 weeks). The early phase involves an initial response to tendon transection, such as bleeding, accumulation of initial extracellular matrix, and an increase in the cell population at the transection site, leading to re-connection of transected tendon stubs, whereas the late phase is dedicated to maturation of regenerated collagenous tissue into new tendon. This is the first study to reveal structural mechanisms of newt tendon regeneration following transection. It warrants further study to explore molecular mechanisms that might achieve such regeneration in mammalian tendon.