Substrate Microarchitecture Shapes the Paracrine Crosstalk of Stem Cells with Endothelial Cells and Osteoblasts

We examined the hypothesis that substrate microarchitecture regulates the crosstalk between human mesenchymal stem cells (hMSC) and cell types involved in bone regeneration. Compared with polyester flat substrates having uniformly distributed homogenous pores (2D), three-dimensional polystyrene substrates with randomly oriented and interconnected pores of heterogeneous size (3D) stimulated the stromal secretion of IGF-1 while lessened the production of VEGFR-1, MCP-1 and IL-6. The medium conditioned by hMSC cultured in 3D substrates stimulated tube formation by human endothelial cells (hEC) to a higher extent than medium from 2D cultures. 3D co-cultures of hMSC and hEC contained higher secreted levels of IGF-1, EGF and FGF-2 than 2D co-cultures, resulting in increased hEC proliferation and migration. Substrate microarchitecture influenced the secretion of factors related to bone remodeling as the ratio RANKL to OPG, and the levels of M-CSF and IL-6 were higher in 3D co-cultures of hMSC and human osteoblasts (hOB) than in 2D co-cultures. Cytokine microenvironment in 3D co-cultures stimulated osteoblast matrix reorganization while demoted the late steps of osteoblastic maturation. Altogether, data in this study may unveil a new role of scaffold microarchitecture during bone regeneration, as modulator of the paracrine relationships that hMSC establish with hEC and hOB.