Ex vivo 3D osteocyte network construction with primary murine bone cells

Abstract

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Tissue engineering: Reproducing bone cell networks in three dimensions

Growing three-dimensional networks from bone cells (osteocytes) is now possible using microbeads as templates. Osteocyte structural cells play crucial roles in regulating bone and mineral balance in the body, but scientists lack suitable models to study their intricate physiological processes.WooLee and coworkers from the Stevens Institute of Technology in New Jersey, USA, harvested cells from mice and generated the osteocyte networks by perfusing calcium phosphate beads. Confined in a culture chamber, the beads formed a porous structure exhibiting evenly distributed spaces that entrapped the cells. The star-shaped cells extended and connected with each other through the spaces between the beads. Unlike two-dimensional cell cultures, the interconnected cells expressed genes typical of mature osteocytes and did not proliferate. This tissue is expected to clarify osteocyte function and provide clinical models for bone diseases.

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