Cellular senescence occurring in the rabbit medial collateral ligament during healing

Medial collateral ligament (MCL) healing proceeds in a temporally ordered fashion after injury. Despite the critical roles of fibroblasts during ligament repair, the phenotypic features of these healing fibroblasts have not been well characterized. Here, we show that healing MCL fibroblasts obtained from rabbits at 3-week postinjury exhibited higher rates of senescent phenotypes and produced higher levels of TGF-β1, collagens, α-SMA, and matrix metalloproteinases (MMPs), than the corresponding fibroblasts from sham-operated MCLs. Mechanical stretch further enhanced the cellular senescence and the expression of TGF-β1, collagens, α-SMA, and MMPs in both sham and healing MCL fibroblasts. In addition to MCL fibroblasts at 3-week postinjury, the increased cellular senescence was also detected in healing MCL fibroblasts obtained at 4- and 6-week postinjury. Most importantly, the association between the cellular senescence and ligament healing was confirmed in tissue sections by the senescence-associated β-galactosidase (SA-β-gal) staining. Using a recombinant TGF-β1 and a neutralizing antibody, we found that those phenotypic changes, such as cellular senescence and the expression of collagens and MMPs, in MCL fibroblasts under mechanical loading conditions were regulated through TGF-β1. Taken together, our results propose that cellular senescence and turnover of extracellular matrixes regulated by TGF-β1 in MCL fibroblasts are critical for ligament healing.