A Mechanically Stimulated Co-culture in 3-Dimensional Composite Scaffolds Promotes Osteogenic and Anti-osteoclastogenic Activity and M2 Macrophage Polarization

Abstract

Bone is subjected to a plethora of mechanical stresses, which have been found to directly influence the equilibrium between bone resorption and formation. Taking this into account, we present herein a novel biomimicking 3-dimensional model that applies cyclic uniaxial compression onto cells co-cultured on 3-dimensionally printed scaffolds consisting of poly L-lactic acid/poly(epsilon-caprolactone)/poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/Sr-nanohydroxyapatite. The aim is to investigate how compression can modulate the balance between osteogenesis and osteoclastogenesis in co-culture, as well as the polarization of macrophages. One of the key aspects of the current study is the unprecedented development of a growth-factor-free co-culture, sustainable solely by the cross talk between human bone marrow mesenchymal stem cells and human peripheral blood mononuclear cells for their survival and osteogenic/osteoclastogenic differentiation capacity, respectively. Real-time polymerase chain reaction gene expression analysis of the mechanically stimulated constructs revealed up-regulation of the osteogenesis-related markers osteocalcin, osteoprotegerin, and runt-related transcription factor 2, with concurrent down-regulation of the osteoclastogenic markers dendritic-cell-specific transmembrane protein, nuclear factor of activated T cells 1, and tartrate acid phosphatase. The secretion of the receptor activator of nuclear factor kappa-Beta ligand and macrophage colony-stimulating factor, as determined from enzyme-linked immunosorbent assay, was also found to depict lower levels compared to static conditions. Finally, macrophage polarization was examined via confocal imaging of tumor necrosis factor-alpha and interleukin-10 secretion levels, as well as through nitric oxide synthase and arginase 1 markers' gene expression, with the results indicating stronger commitment toward the M2 phenotype after mechanical stimulation.