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.

Preclinical models for in vitro mechanical loading of bone-derived cells

“…Typical (2)) that is oscillatory in nature (c) is produced across the well bottom (x/L is a point in space along the well bottom, where x/L ¼ 0.5 is the center) at different points in the cycle. [26][27][28] Matrix production is enhanced by 'see-saw' rocking in osteogenic progenitor cells. (d) Second harmonic generation imaging shows increased collagen deposition/organization by human dermal fibroblasts (hDFs) and human embryonic stem mesenchymal progenitors (hES-MPs) subjected to OFSS versus static (no-flow) conditions.…”

“…Until recently, none of the available 3D in vitro bone models investigated osteocyte- This has recently been overcome via a new 3D in vitro osteocyte-osteoblast coculture mechanical loading model, cultured within a custom-built multiwell silicone loading plate, using MLO-Y4 osteocyte-like cells and MC3T3-E1 (14) or MG63 osteoblast like cells. 26 In this model, MLO-Y4 cells were incorporated within rat tail tendon type I collagen gels (2-2.6 mg ml À 1 ) and either MC3T3-E1 (14) or MG63 cells layered on the top. MLO-Y4 cells (1.5 Â 10 6 cells per ml gel) diluted in the culture medium (o10% of total gel volume) were embedded in the collagen and distributed into multiwell plates for polymerization (37 1C, 1 h).…”

“…MC3T3-E1 (14) or MG63 cells (1.5 Â 10 5 cells per well) in the culture medium were then applied onto the surface of the gel incubated at 37 1C (Figure 6a). 26 The ratio of osteocytes and osteoblasts was 10:1 to mimic in vivo bone where there are 10 times more osteocytes than osteoblasts. 76 The cells within the model were viable, maintained typical osteocyte morphology and phenotype and appeared to connect to neighboring cells.…”

“…The system was validated using Digital Image Correlation, which revealed strains of 4000-4500 me when a force of 2.5 N was applied to the silicone plate. 26 Currently, there are two published devices that are similar to the one developed by Vazquez et al 77,78 These are a six-well silicone plate used to mechanically load vascular smooth muscle cells in monolayers, 78 and a single-well silicone plate designed to load 3D collagen cultures of intervertebral disc cells. 77 Both devices applied cyclic mechanical stimuli in a similar manner to the device described by Vazquez et al, but instead used higher strains and lower frequencies (24 h, 0.1 Hz, 10 000 me; 77 6-72 h, 1 Hz, 10-20% strain).…”

“…Furthermore, coculture models of osteoblasts and osteocytes have been developed, which will allow for the interaction between these important bone cell types to be studied in response to loading. 26 This technical review will describe optimized techniques for loading bone-derived cells by vibration, fluid flow, substrate strain or compression in monolayer and 3D cultures to provide a concise yet comprehensive guide to bone cell loading in vitro.…”