title: A new method to investigate how mechanical loading of osteocytes controls osteoblasts.
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.
authors.affiliation: Arthritis Research UK Biomechanics and Bioengineering Centre, School of Biosciences, Cardiff University , Cardiff , UK.
authors.forename: Marisol
authors.initials: M
authors.lastname: Vazquez
authors.affiliation: Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University , Cardiff , UK.
authors.forename: Bronwen A J
authors.initials: BA
authors.lastname: Evans
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Cardiff University , Cardiff , UK.
authors.forename: Daniela
authors.initials: D
authors.lastname: Riccardi
authors.affiliation: Institute of Mechanical and Manufacturing Engineering, School of Engineering, Cardiff University , Cardiff , UK.
authors.forename: Sam L
authors.initials: SL
authors.lastname: Evans
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Cardiff University , Cardiff , UK.
authors.forename: Jim R
authors.initials: JR
authors.lastname: Ralphs
authors.affiliation: School of Psychology, Cardiff University , Cardiff , UK.
authors.forename: Christopher Mark
authors.initials: CM
authors.lastname: Dillingham
authors.affiliation: Division of Pathophysiology and Repair, School of Biosciences, Cardiff University , Cardiff , UK.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Mason
keywords: 3 dimensional; co-culture; loading; model; osteoblast; osteocyte