Multimaterial Triphasic Scaffold Replicates Enthesis Microenvironment to Modulate Early Cell Responses

Current surgical approaches to reconstruct functional organization of tendon/ligament‐to‐bone interface, namely enthesis, often fail, highlighting the need for bioinspired tissue engineering strategies that replicate its native complexity. This study investigates the cellular response to a triphasic scaffold designed to mimic the enthesis gradient organization. The scaffold combines a fused deposition modelling polycaprolactone region mimicking bone, an electrospun poly(lactic‐co‐glycolic acid) (PLGA) region as tendon, with an overlap between the two to replicate enthesis. Human mesenchymal stem cells are seeded onto the scaffold, and their proliferation and morphology are assessed under static and mechanically stimulated conditions (two stimulation protocols) using a custom‐designed bioreactor. Compared to static, in the first dynamic condition, cells align and elongate more along PLGA fibers, whilst the second protocol results in lesser elongation. Gene expression analysis shows moderate, but lineage‐specific expression of tendon (SCXA, EGR1/2), cartilage (SOX9, ACAN), and bone (RUNX2, IBSP) markers. Tendon‐related THBS2 is upregulated in the fibrocartilage‐ and tendon‐like regions; moreover, the tendon maturation marker tenomodulin shows moderate expression in bone and tendon‐like zones and upregulates under load in the fibrocartilage zone. These results demonstrate the potential of cyclic strain stimulation combined with the triphasic scaffold to spatially define cellular behaviors for enthesis tissue engineering.