Microbeads-Guided Reconstruction of 3D Osteocyte Network during Microfluidic Perfusion Culture

Osteocytes reside as 3-dimensionally networked cells in the lacunocanalicular structure of bones, and function as the master regulators of homeostatic bone remodeling. We report here, for the first time to our best knowledge, the use of a biomimetic approach to reconstruct the 3D osteocyte network with physiological relevant microscale dimensions. In this approach, biphasic calcium phosphate microbeads were assembled with murine early osteocytes (MLO-A5) to provide an initial mechanical framework for 3D network formation and maintenance during long-term perfusion culture in a microfluidic chamber. The microbead size of 20-25 μm was used to: (1) facilitate a single cell to be placed within the interstitial space between the microbeads, (2) mitigate the proliferation of the entrapped cell due to its physical confinement in the interstitial site, and (3) control cell-to-cell distance to be 20-25 μm as observed in murine bones. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads within 3 days of culture. The entrapped cells produced significant mineralized extracellular matrix to fill up the interstitial spaces, resulting in the formation of a dense tissue structure during the course of 3-week culture. We found that the time-dependent osteocytic transitions of the cells exhibited trends consistent with in vivo observations, particularly with high expression of Sost gene, which is a key osteocyte-specific marker for the mechanotransduction function of osteocytes. In contrast, cells cultured in 2D well-plates did not replicate in vivo trends. These results provide an important new insight in building physiologically relevant in vitro bone tissue models.