Spatially Regulated Multiphenotypic Differentiation of Stem Cells in 3D via Engineered Mechanical Gradient

Within the osteochondral interface, cellular and extracellular matrix gradients provide a biomechanical and biochemical niche for homeostatic tissue functions. Postnatal joint loading is critical for the development of such tissue gradients, leading to the formation of functional osteochondral tissues composed of superficial, middle, and deep zones of cartilage, and underlying subchondral bone, in a depth-dependent manner. In this regard, a novel, variable core-shell electrospinning strategy was employed to generate spatially controlled strain gradients within three-dimensional scaffolds under dynamic compressive loading, enabling the local strain-magnitude dependent, multiphenotypic stem cell differentiation. Human mesenchymal stem cells (hMSCs) were cultured in electrospun scaffolds with a linear or biphasic mechanical gradient, which was computationally engineered and experimentally validated. The cell/scaffold constructs were subjected to various magnitudes of dynamic compressive strains in a scaffold depth-dependent manner at a frequency of 1 Hz for 2 h daily for up to 42 days in osteogenic media. Spatially upregulated gene expression of chondrogenic markers (ACAN, COL2A1, PRG4) and glycosaminoglycan deposition was observed in the areas of greater compressive strains. In contrast, osteogenic markers (COL1A1, SPARC, RUNX2) and calcium deposition were downregulated in response to high local compressive strains. Dynamic mechanical analysis showed the maintenance of the engineered mechanical gradients only under dynamic culture conditions, confirming the potent role of biomechanical gradients in developing and maintaining a tissue gradient. These results demonstrate that multiphenotypic differentiation of hMSCs can be controlled by regulating local mechanical microenvironments, providing a novel strategy to recapitulate the gradient structure in osteochondral tissues for successful regeneration of damaged joints in vivo and facile development of interfacial tissue models in vitro.