mTORC1 and STAT3 signaling are indispensable for in vitro TGFβ1-dependent three-dimensional (3D) tendon constructs.

Transforming Growth Factor-β1 (TGFβ1) is an established growth factor that regulates tenocyte differentiation, extracellular matrix production, and cell fate. We previously demonstrated its pivotal role in in vitro 3D tendon constructs formation; however, the downstream signaling mechanisms remain elusive. In this study, we explore the roles of mTORC1 (Mammalian Target of Rapamycin Complex 1) and STAT3 (Signal Transducer and Activator of Transcription 3) in mediating TGFβ1-induced 3D tendon formation using rapamycin (an mTORC1 inhibitor) and stattic (a STAT3 inhibitor). Inhibition of either pathway compromised TGFβ1-induced thickening of the tendon construct, cellular proliferation, and collagen fibrillogenesis. Molecular analyses revealed that mTORC1-STAT3 signaling partially mediates TGFβ1-induced Scx expression and tenocyte elongation in the peripheral layer of 3D tendon constructs. Moreover, TGFβ1 treatment augmented mTOR and STAT3 phosphorylation, while inhibition of mTORC1 signaling attenuated TGFβ1-induced STAT3 phosphorylation. These findings underscore the TGFb1-mTORC1- STAT3 signaling pathway as integral to 3D tendon constructs. Overall, our study identifies the mTORC1-STAT3 axis as a crucial mediator of TGFβ1-driven in vitro tendon formation, highlighting its importance in tendon maturation and extracellular matrix organization.