Extrusion Printed Scaffolds with Varying Pore Size As Modulators of MSC Angiogenic Paracrine Effects

Cell encapsulation in confining 3D hydrogels typically prevents encapsulated cells from spreading and establishing cell-cell contacts. Interactions with neighboring cells or with the extracellular matrix (ECM) influence the paracrine activity of mesenchymal stromal cells (MSCs), but how these interactions are regulated by structural properties of biomaterial scaffolds remains insufficiently explored. Here, we describe the use of extrusion-based 3D printing to fabricate acellular, gelatin-based scaffolds with programmed strut spacings of 400 (small), 500 (medium), and 600 μm (large). These scaffolds showed similar effective Young's moduli in the range of 2-5 kPa, and varied based on average pore size which ranged from ∼200 μm (small pore: SP) through ∼302 μm (medium pore: MP) to ∼382 μm (large pore: LP). When seeded with MSCs, pore size guided cell distribution on the scaffolds, with smaller pores preventing cell infiltration, medium ones causing cells to aggregate in between struts, and large ones causing cells to flow through after attachment on the struts. These changes in cell distribution regulated cell-cell and cell-matrix interactions at the gene level, as assessed by pathway focused PCR arrays. Medium pore size scaffolds stimulated the highest paracrine secretion of a panel of angiogenic cytokines. This enhancement of paracrine activity substantially improved endothelial cell migration in a chemotaxis assay, increased single cell migration kinetics such as velocity, and stimulated the formation of robust tubular structures. Together, these findings not only provide new insights on cellular interactions in scaffold environments but also demonstrate how 3D biomaterial design can instruct and enhance the regenerative paracrine activities of MSCs.