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

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