How the Hydrogel Scaffold’s Porous Structure and Composition Control the Formation of Spheroids for Bone Tissue Engineering

When spheroids are used as building blocks in scaffold-based tissue regeneration strategies, two successive steps are typically involved: the fabrication of the spheroids using dedicated techniques and their incorporation into the scaffold. Here, we studied a scaffold-based approach involving a non-cell-adhesive porous biomaterial, where the cell suspension is seeded directly onto the scaffold, spontaneously forming spheroids within its pores. This “two-step-in-one” route is illustrated with a cross-linked pullulan/dextran hydrogel supplemented with hydroxyapatite (HA) particles (6% w/w in the dry state), entirely dispersed within the hydrogel phase, and textured by freeze-drying. Pores and spheroids were characterized in 3D using state-of-the-art tomographic techniques. The method was applied to the formation and in vitro coculture of MSCs/HUVECs spheroids for vascularized bone tissue engineering. Cells suspended in culture medium were seeded onto the hydrogel scaffold initially in its dry state, and the gel swelled concomitantly with seeding. The scaffold porosity decreased from 80% to 25%, the mean pore size decreased from 350 to 180 μm, and the pores underwent a buckling transition, evolving from spherical to lenticular (sphericity decreased from 0.71 to 0.49). At the same time, the cells condensed into grape-like aggregates, accelerated by two mechanisms: increased local cell density resulting from suction of the culture medium by the dry gel and geometric confinement imposed by shrinking pores. Then, the cell aggregates compacted with a characteristic time of about 3 h, transitioning from a lenticular shape inherited from the pores to a spheroidal shape (mean size of 130 μm, mean sphericity of 0.82). Later, the cellularized HA-supplemented hydrogel scaffolds induced early phase bone differentiation (ALP peaks at day 7), whereas pristine hydrogel scaffolds did not, although cells were cultured in a calcifying medium. Further osteogenic maturation was observed for spheroids cocultured in HA-supplemented scaffolds under dynamic conditions, which exhibited mineralized extracellular matrix. This led us to re-examine the physical mechanisms underlying HA’s osteoinductive properties. Together, these findings inform scaffold-based tissue-engineering strategies and open an alternative way for the high-throughput production of mature spheroids.