Microbead-guided reconstruction of the 3D osteocyte network during microfluidic perfusion culture

Abstract

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.

Ex vivo construction of human primary 3D–networked osteocytes

“…2d). The 3D culture results obtained for the human cells were similar to those observed with primary murine bone cells[32] and MLO-A5[34] except for that MLO-A5 produced significantly more ECM than the primary cells, and therefore a much denser tissue structure. [34]…”

“…The cell-to-cell distance was in part controlled by the diameter of the microbeads, as discussed in our prior publications. [32], [34] Cell proliferation was inhibited within the interstitial confine between the microbeads.…”

“…10 µL of the mixture was placed into the culture chamber using a micropipette. Cells and microbeads were assembled with details described in our previous study[34], and as illustrated in Fig. 1b.…”

“…We used a microbeads-guided assembly approach that: (1) consists of 3D cellular network of primary osteocytes, and (2) mitigate the rapid osteoblastic dedifferentiation and proliferation of primary osteoblast-like encountered in conventional 2D culture. [32], [33] In this biomimetic assembly approach[32], [34]and as illustrated in Fig. 1b, biphasic calcium phosphate (BCP) microbeads of 20–25 µm were used to: (1) spatially distribute osteocyte cell bodies into the interstitial spaces between the microbeads with one cell occupying each interstitial site while allowing them to develop processes with neighboring cells with the physiologically relevant lacuna and interlacunar dimensions and (2) provide a mechanically stable framework to maintain the microscale geometry and dimensions of the 3D cellular network during perfusion culture.…”

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes

“…Celllines could lead to different results with respect to primary cells from the same species. For example, murine early osteocyte cell-line MLO-A5 created an interconnected 3D network in a microfluidic perfusion device (Gu et al, 2015) but produced a highly mineralized and dense tissue compared to primary murine bone cells (Sun et al, 2015).…”