In vitro 3D osteoblast-osteocyte co-culture mechanical loading model

Vazquez, Marisol; Evans, Bronwen Alice James; Evans, S.C; Ralphs, J.R.; Riccardi, Daniela; Mason, Deborah Jane

doi:10.1530/boneabs.1.pp207

Cited by 3 publications

(3 citation statements)

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“…This hypoxia-induced self-assembly of the endosteum-like layer is particularly noteworthy, as it has not been observed in any other culture system reported to date. [30][31][32]37,38 Second, hypoxia significantly enhanced the gene expressions of SOST and FGF23, while decreasing that of ALPL. Taking culture conditions separately, replacing traditional normoxic 2D cultures with hypoxia significantly increased SOST expression by fourfold, while introducing the 3D configuration to the traditional 2D normoxic cultures significantly increased SOST by twofold (Fig.…”

Section: Discussionmentioning

confidence: 95%

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

Choudhary

Sun

Mannion

et al. 2018

Tissue Engineering Part A

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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.

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Section: Discussionmentioning

confidence: 95%

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

Choudhary

Sun

Mannion

et al. 2018

Tissue Engineering Part A

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“…Although some coculture models have been established to research the direct (physical) intercellular communication in mechanotransduction, they still have a lot of limitations due to osteocytes' special morphological characteristics and special location in bone. The trabecular bone explant model and the 3D osteoblast-osteocyte coculture model could simulate in vivo intercellular communication between embedded osteocytes and surface-residing osteoblasts very well (Mitsiades et al 2000;Chan et al 2009;Vazquez et al 2011Vazquez et al , 2012. However, they are difficult not only to operate but also to quantitatively research, as well as it is not easy to retrieve osteocytes and osteoblasts respectively for the next experiments.…”

Section: Discussionmentioning

confidence: 99%

Gap junction mediated regulation of osteocytes to osteoblastic alkaline phosphatase activity is independent of microgravity

Meng

Xie

2014

Animal Cells and Systems

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Decreased bone formation is one of the main causes of bone loss under microgravity. As a mechanical perceiver, osteocyte regulates bone formation by sending mechanical signals to osteoblast. However, the regulation of osteoblastic bone formation by osteocytes is less known so far under the microgravity conditions. The aim of this study was to investigate the regulation of bone formation/loss by detection of the osteocytic regulatory effects on the bone-specific alkaline phosphatase (ALP) activity of osteoblasts under altered gravitational environment. The altered gravitational environment was provided by a large gradient high magnetic field that could produce high magneto-gravitational environment (HMGE) and provided three apparent gravity levels (µg, 1g, and 2g). After the MLO-Y4 and 2T3 cells were cocultured in direct physical contact for 24 h and established gap junction intercellular communication under HMGE, a highly significant rapid increase in ALP activity was observed in µg, 1g, 2g, and control group (p < 0.001). Conversely, application of 50-µM beta-glycyrrhetinic acid gap junction inhibitor or remote coculture significantly decreased the ALP activity. There were no obvious differences of osteoblastic 2T3 ALP activity among µg, 1g, 2g, and control groups. Based on these findings, we interpreted that gap junction was probably the main route of osteoblastic ALP activity regulation by osteocyte. Gravity had no significant impact on the osteoblastic ALP activity regulated by osteocyte in a short period of time.

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“…More precisely, several experiments using conditioned medium (i.e. supernatant containing soluble factors from osteocytes) (155,156) or different co-culture methodologies (155,157,158), in 2D substrate or in 3D gels (158,159), support the concept that osteocytes regulate the function of both osteoblasts and osteoclasts. Moreover, considerable convincing data emphasize the role of osteocytes as mechanosensors, integrating mechanical signals to regulate bone mass (153).…”

Section: Complex 3d Models: Cell Co-culturementioning

confidence: 94%

In VitroThree-Dimensional Bone Tissue Models: From Cells to Controlled and Dynamic Environment

Bouet

Marchat

Cruel

et al. 2015

Tissue Engineering Part B: Reviews

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Most of our knowledge of bone cell physiology is derived from experiments carried out in vitro on polystyrene substrates. However, these traditional monolayer cell cultures do not reproduce the complex and dynamic three-dimensional (3D) environment experienced by cells in vivo. Thus, there is a growing interest in the use of 3D culture systems as tools for understanding bone biology. These in-vitro-engineered systems, less complex than in vivo models, should ultimately recapitulate and control the main biophysical, biochemical, and biomechanical cues that define the in vivo bone environment, while allowing their monitoring. This review focuses on state-of-the-art and the current advances in the development of 3D culture systems for bone biology research. It describes more specifically advantages related to the use of such systems, and details main characteristics and challenges associated with its three main components, that is, scaffold, cells, and perfusion bioreactor systems. Finally, future challenges for noninvasive imaging technologies are addressed.

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In vitro 3D osteoblast-osteocyte co-culture mechanical loading model

Cited by 3 publications

References 0 publications

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

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

Gap junction mediated regulation of osteocytes to osteoblastic alkaline phosphatase activity is independent of microgravity

In VitroThree-Dimensional Bone Tissue Models: From Cells to Controlled and Dynamic Environment

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