Mechanical activation drives tenogenic differentiation of human mesenchymal stem cells in aligned dense collagen hydrogels

This study used gel aspiration-ejection (GAE) to rapidly fabricate anisotropic aligned dense collagen (ADC) hydrogels (5–13 wt% collagen) with tendon-like alignment and stiffness. Acellular ADCs tolerated short-term high-strain mechanoactivation (48 h at 20% strain) and showed increased collagen fibril thickness and improved tensile properties, including ~2.7–3.0× increases in ultimate tensile strength and apparent modulus after static strain and a significant modulus increase after cyclic-rest loading. Human bone marrow MSCs were 3D-embedded in ADCs and mechanically activated for 48 h under serum- and growth factor-free conditions; both static and cyclic-rest profiles drove strong tenogenic commitment (>12× scleraxis upregulation) while suppressing or maintaining osteogenic (RUNX2) and chondrogenic (aggrecan) markers compared with controls. Following mechanoactivation, constructs were matured for 19 additional days without external loading, resulting in polarized, aligned cells and deposition of tendon-associated matrix proteins (COL I/III/VI, tenascin-C, tenomodulin), with cyclic-rest stimulation producing markedly higher tenomodulin protein levels, indicating loading-profile-dependent maturation toward a tendon-like phenotype.