# An osteoblast-like cell line derived from mice expressing FRET-based tension sensor reveals cellular tension increase during osteogenic differentiation

## TL;DR

Researchers created a new cell line that can measure internal cell tension in real time, revealing that tension increases during bone cell development.

## Contribution

A novel osteoblast-like cell line with a FRET-based tension sensor for real-time intracellular tension monitoring during osteogenic differentiation.

## Key findings

- The FRET1-MC8 cell line showed stable proliferation and mineralization up to passage 65.
- Osteogenic induction increased intracellular tension and Spp1 gene expression.
- The FRET-based sensor enabled non-invasive real-time tension measurement in live cells.

## Abstract

Mechanical stimuli significantly influence bone remodeling, although the detailed molecular mechanisms involving changes in intracellular tension during osteoblast differentiation remain unclear. The present study was performed to investigate the role of intracellular tension in osteogenic differentiation by utilizing a newly established osteoblast-like cell line. In this study, we established a novel osteoblast-like cell line derived from calvarial explants of transgenic mice ubiquitously expressing a Förster Resonance Energy Transfer (FRET)-based tension sensor, capable of real-time measurement of intracellular tension and mineralization. The established cell line FRET1-MC8 displayed superior proliferative ability compared to conventional MC3T3-E1 osteoblast-like cells, maintaining stable growth and mineralization capability through at least passage 65. Osteogenic medium (OM) significantly enhanced the expression of osteogenic differentiation markers Col1a1 and Spp1, with clear mineralization observed as early as day 7 and extensive mineralization by day 14, comparable to MC3T3-E1 cells. Scratch experiments revealed increase in apparent contractility in OM-cultured cells, and subsequent quantitative analysis using acceptor photobleaching-based FRET efficiency confirmed significantly increased intracellular tension at day 3 of osteogenic induction. This elevation in intracellular tension coincided with increased Spp1 expression, suggesting a critical role of tension in promoting osteogenic differentiation. The use of a FRET-based tension sensor enabled fast and direct monitoring of intracellular tension and is applicable to real-time analysis using live-cell FRET imaging. The established FRET1-MC8 cell line provides a powerful research tool for direct measurement of intracellular tension during osteogenic differentiation, thereby contributing to a better understanding of the mechanical regulation of bone remodeling.

•Novel osteoblastic cell line expressing a FRET-based tension sensor was established.•The cells showed stable proliferation and mineralization capability over passage 65.•Osteogenic induction increased intracellular tension and Spp1 gene expression.•FRET-based sensor may enable non-invasive real-time measurement of cellular tension.•It is a useful tool for investigating mechanical regulation in bone remodeling.

Novel osteoblastic cell line expressing a FRET-based tension sensor was established.

The cells showed stable proliferation and mineralization capability over passage 65.

Osteogenic induction increased intracellular tension and Spp1 gene expression.

FRET-based sensor may enable non-invasive real-time measurement of cellular tension.

It is a useful tool for investigating mechanical regulation in bone remodeling.