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A new method to investigate how mechanical loading of osteocytes controls osteoblasts

Vazquez, Marisol, Evans, Bronwen Alice James, Riccardi, Daniela, Evans, Samuel Lewin, Ralphs, James Robert, Dillingham, Christopher Mark and Mason, Deborah Jane 2014. A new method to investigate how mechanical loading of osteocytes controls osteoblasts. Frontiers in Endocrinology 5 , 208. 10.3389/fendo.2014.00208

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Abstract

Mechanical loading, a potent stimulator of bone formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) in vitro co-culture system to investigate the effect of loading on osteocyte–osteoblast interactions. MLO-Y4 cells were embedded in type I collagen gels and MC3T3-E1(14) or MG63 cells layered on top. Ethidium homodimer staining of 3D co-cultures showed 100% osteoblasts and 86% osteocytes were viable after 7 days. Microscopy revealed osteoblasts and osteocytes maintain their respective ovoid/pyriform and dendritic morphologies in 3D co-cultures. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of messenger ribonucleic acid (mRNA) extracted separately from osteoblasts and osteocytes, showed that podoplanin (E11), osteocalcin, and runt-related transcription factor 2 mRNAs were expressed in both cell types. Type I collagen (Col1a1) mRNA expression was higher in osteoblasts (P < 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (P = 0.001). Immunohistochemistry revealed osteoblasts and osteocytes express E11, type I pro-collagen, and connexin 43 proteins. In preliminary experiments to assess osteogenic responses, co-cultures were treated with human recombinant bone morphogenetic protein 2 (BMP-2) or mechanical loading using a custom built loading device. BMP-2 treatment significantly increased osteoblast Col1a1 mRNA synthesis (P = 0.031) in MLO-Y4/MG63 co-cultures after 5 days treatment. A 16-well silicone plate, loaded (5 min, 10 Hz, 2.5 N) to induce 4000–4500 με cyclic compression within gels increased prostaglandin E2 (PGE2) release 0.5 h post-load in MLO-Y4 cells pre-cultured in 3D collagen gels for 48, 72 h, or 7 days. Mechanical loading of 3D co-cultures increased type I pro-collagen release 1 and 5 days later. These methods reveal a new osteocyte–osteoblast co-culture model that may be useful for investigating mechanically induced osteocyte control of osteoblast bone formation.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Biosciences
Engineering
Medicine
Psychology
Subjects: B Philosophy. Psychology. Religion > BF Psychology
Q Science > QR Microbiology
R Medicine > R Medicine (General)
T Technology > TJ Mechanical engineering and machinery
Publisher: Frontiers Research Foundation
ISSN: 1664-2392
Funders: Arthritis Research UK
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 18 November 2014
Last Modified: 03 May 2019 14:09
URI: http://orca.cf.ac.uk/id/eprint/69061

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