Hypoxic Three-Dimensional Cellular Network Construction ReplicatesEx Vivothe Phenotype of Primary Human Osteocytes

Abstract

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.

A 3D, Dynamically Loaded Hydrogel Model of the Osteochondral Unit to Study Osteocyte Mechanobiology

“…researchers developed 3D osteocyte culture models to improve upon the limitations of 2D cultures. [27][28][29][30][31][32][33] For example, one study achieved mature osteocyte differentiation in a 3D collagen gel but this culture system lacked mechanical cues. [34] Other studies revealed new effects of direct matrix strains on osteocytes in 3D [27,29,35] such as strain-induced osteocyte regulation of osteoblast bone formation.…”

“…[27] These prior studies applied matrix strains ranging from 0.4-10%, which exceed physiological levels of strain in bone. Other studies incorporated fluid perfusion into 3D cultures [15,28,30,32,36,37] and revealed, for example, the link between fluid-induced shear and gene expression of proteins that regulate osteoblast bone formation (e.g., sclerostin). [28] However, 3D models that adequately capture both physiologically relevant matrix strains and fluid perfusion are limited.…”

Human ex vivo 3D bone model recapitulates osteocyte response to metastatic prostate cancer

“…1) by culturing primary human osteocytes with BCP microbeads for 14 days. Comparisons of dendrite length and cell-cell distance (Table 1) 40 between hypoxic and normoxic (from our previous studies 38 ) 3D bone tissues revealed that hypoxia induces profound changes in the 3D structure of osteocytic cells by producing cells with prolonged dendrites.

Figure 1Microfluidic perfusion device for engineering 3D bone tissues. ( a ) Actual device, containing bone tissue constructs in the central chamber with medium flowing into one inlet fed by a syringe pump and exiting through two outlets carrying effluent to a collection vial.

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“…In the tissue without PCa cells (−PCa cells, Fig. 2a), the osteocytes were well spread out, with dendrites protruding to neighboring cells (inset) and the endosteal layer (characterized previously 38 ) was intact (Fig. 1a, black arrows).

Figure 2Histology sections of the engineered 3D bone tissues.

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“…Specifically, we showed that primary human osteocytes can be assembled with 20–25 µm microbeads and cultured in a microfluidic perfusion device to replicate the lacunocanalicular structure and functions of human bone tissue 35,37 . Most recently, we established that hypoxic 3D culture of human primary osteocytic cells enhanced osteocyte phenotype ex vivo while enabling the spontaneous formation of an osteoblastic monolayer that resembles the endosteal layer 38 . This single cell layer primarily comprised of osteoblasts and localized at the interface between the bone marrow and bone, is critical in bone metastasis since it represent the site where disseminated tumor cells interact with the bone and become dormant and drug resistance until tumor reactivation and progression 39 .…”