Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests that osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption; however, the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using three-dimensional (3D) culture substrates in OCY454 cells suggest that 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and receptor activator of nuclear factor-κB ligand (RANKL). In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds with traditional 2D tissue culture, both with [wild-type (WT)] and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures were used to determine soluble signaling to differentiate primary bone marrow cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared with that of Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion.NEW & NOTEWORTHY 3D cell culture of OCY454 cells promoted increased differentiation compared with traditional 2D culture. Although Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.
Keywords: 3D cell culture; connexin 43; gap junctions; in vitro; osteocytes.