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

Vazquez, Marisol, Evans, Bronwen A. J. ORCID:, Riccardi, Daniela ORCID:, Evans, Sam L. ORCID:, Ralphs, Jim R. ORCID:, Dillingham, Christopher Mark and Mason, Deborah J. ORCID: 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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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: Publication
Status: Published
Schools: Biosciences
Subjects: B Philosophy. Psychology. Religion > BF Psychology
Q Science > QR Microbiology
R Medicine > R Medicine (General)
T Technology > TJ Mechanical engineering and machinery
Additional Information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).
Publisher: Frontiers Media
ISSN: 1664-2392
Funders: Arthritis Research UK
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 18 November 2014
Last Modified: 05 Jan 2024 02:38

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