Niche-mimicking interactions in peptide-functionalized 3D hydrogels amplify mesenchymal stromal cell paracrine effects

Cells encounter complex environments in vivo where they interact with the extracellular matrix, neighboring cells, and soluble cues, which together influence their fate and function. However, the interplay of these interactions and their collective impact on the regenerative effects of mesenchymal stromal cells (MSCs) remains insufficiently explored. Here, we show that 3D culture in microporous (~125 μm) hydrogels that passively promote cell-cell interactions sensitizes MSCs to growth factors, particularly to IGF-1. IGF-1 enhances MSC paracrine secretion activity, and application of secreted factors to myoblasts potently stimulates their migration and differentiation. In contrast, the paracrine activity of MSCs encapsulated in nanoporous (~10 nm) hydrogels remain unchanged. Blocking N-cadherin on MSCs abrogates the stimulatory effects of IGF-1 in microporous but not nanoporous hydrogels. The role of N-cadherin in regulating MSC function is further clarified by functionalizing alginates with the HAVDI peptide sequence that is derived from the extracellular domain of N-cadherin and that acts to mimic cell-cell interactions. MSCs encapsulated in nanoporous HAVDI-gels, but not in gels functionalized with a scrambled sequence, show heightened paracrine activity in response to IGF-1. These findings reveal how interactions with the matrix, neighboring cells, and soluble factors impact and maximize the regenerative potential of MSCs.