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

Marisol Vazquez; Bronwen A J Evans; Daniela Riccardi; Sam L Evans; Jim R Ralphs; Christopher Mark Dillingham; Deborah J Mason

doi:10.3389/fendo.2014.00208

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

Front Endocrinol (Lausanne). 2014 Dec 9:5:208. doi: 10.3389/fendo.2014.00208. eCollection 2014.

Authors

Marisol Vazquez¹, Bronwen A J Evans², Daniela Riccardi³, Sam L Evans⁴, Jim R Ralphs³, Christopher Mark Dillingham⁵, Deborah J Mason³

Affiliations

¹ Arthritis Research UK Biomechanics and Bioengineering Centre, School of Biosciences, Cardiff University , Cardiff , UK.
² Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University , Cardiff , UK.
³ Division of Pathophysiology and Repair, School of Biosciences, Cardiff University , Cardiff , UK.
⁴ Institute of Mechanical and Manufacturing Engineering, School of Engineering, Cardiff University , Cardiff , UK.
⁵ School of Psychology, Cardiff University , Cardiff , UK.

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.

Keywords: 3 dimensional; co-culture; loading; model; osteoblast; osteocyte.