AUTHOR=Vazquez Marisol , Evans Bronwen A. J. , Riccardi Daniela , Evans Sam L. , Ralphs Jim R. , Dillingham Christopher Mark , Mason Deborah J. 

TITLE=A New Method to Investigate How Mechanical Loading of Osteocytes Controls Osteoblasts

JOURNAL=Frontiers in Endocrinology

VOLUME=5

YEAR=2014

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2014.00208

DOI=10.3389/fendo.2014.00208

ISSN=1664-2392

ABSTRACT=<p>Mechanical loading, a potent stimulator of bone formation, is governed by osteocyte regulation of osteoblasts. We developed a three-dimensional (3D) <italic>in vitro</italic> 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 (<italic>Col1a1</italic>) mRNA expression was higher in osteoblasts (<italic>P</italic> &lt; 0.001), whereas, alkaline phosphatase mRNA was higher in osteocytes (<italic>P</italic> = 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 <italic>Col1a1</italic> mRNA synthesis (<italic>P</italic> = 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 E<sub>2</sub> (PGE<sub>2</sub>) 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.</p>