Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.
The tensile and stress‐rupture behavior of SiC/SiC minicomposite containing a chemically vapor deposited (CVD) ZrO2 interphase was evaluated. Fractographic analyses showed that in situ fiber strength and minicomposite failure loads were strongly dependent on the phase contents and microstructure of the ZrO2 interphase. When the ZrO2 interphase structure possessed a weakly bonded interface within the dense ZrO2 interphase coating layer, the interphase sufficiently protected the fiber surface from processing degradation and promoted matrix crack deflection around the fibers. With this weakly bonded interphase, the stress‐rupture properties of SiC/SiC minicomposite at 950° and 1200°C appeared to be controlled by fiber rupture properties, and compared favorably to those previously measured for state‐of‐the‐art BN fiber coatings.
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