Extracellular Matrix Microspheres with Magnetically Labeled MSCs enable Functional Regeneration of the Osteo-Tendinous Junction

The osteotendinous junction, or enthesis, is a mechanically graded transitional zone critical for load transfer between soft and hard tissues. Its regeneration following injury remains a major clinical challenge due to poor integration of current grafts, uncontrolled alignment of cells, and the lack of spatiotemporal control over cell fate. To address this, we engineered a magneto-responsive, biomimetic system that synergistically combines adipose-derived mesenchymal stem cells (AdMSCs), tendon-derived extracellular matrix (tECM) microspheres, and iron-doped nano magnetized hydroxyapatite (nMHAp) nanoparticles. The nMHAp particles were synthesized via an accelerated biomineralization route, yielding superparamagnetic, osteoinductive particles suitable for intracellular uptake and magnetic manipulation. The tECM microspheres provided a natural tenogenic niche, supporting spatial compartmentalization within a single construct. Upon exposure to a static magnetic field, nMHAp-labeled AdMSCs encapsulated within tECM microspheres exhibited enhanced osteogenic commitment, characterized by a ∼2-fold upregulation of RUNX2 expression and a ∼2-fold increase in mineral deposition compared to nonstimulated controls. Additionally, cells displayed pronounced cytoskeletal alignment under magnetic stimulation. In contrast, in the absence of magnetic cues, the tECM microenvironment preserved tenogenic characteristics, thereby enabling spatially regulated dual-lineage differentiation. Collectively, this multicue strategy provides a dynamic and tunable platform for recapitulating the osteotendinous interface and represents a promising approach for functional enthesis regeneration and complex interface tissue engineering.