# Mitochondrial Imaging and Transcriptome Analysis of Bone Mesenchymal Stem Cells During Osteogenesis Under Different Culture Conditions

## TL;DR

This study compares how bone stem cells behave in 2D and 3D environments during bone formation, focusing on mitochondria and gene activity.

## Contribution

The study reveals how 3D culture conditions influence mitochondrial structure and gene expression during osteogenesis.

## Key findings

- Mitochondria in BMSCs become elongated and interconnected over time during osteogenesis.
- 3D culture systems upregulate genes related to bone development and extracellular matrix organization.
- 3D culture mimics in vivo osteogenesis pathways, suggesting its potential for bone regeneration strategies.

## Abstract

Bone mesenchymal stem cells (BMSCs) are multipotent progenitors with significant potential for bone tissue engineering and regenerative medicine. This study compared the mitochondrial imaging and transcriptome of BMSCs under two-dimensional (2D) and three-dimensional (3D) culture conditions during osteogenesis. 2D BMSCs were induced toward osteogenesis for 7, 14, and 21 days, while 3D BMSCs were induced for 21 days. Osteogenic mineralization was assessed by Alizarin Red S (ARS) staining, and whole-transcriptome sequencing (RNA-Seq) was performed to elucidate gene expression profiles. Furthermore, mitochondrial morphology in live cells was monitored at 0, 7, 14, and 21 days of 2D osteogenic differentiation to observe the mitochondrial changes. High-Sensitivity Structured Illumination Microscopy (HIS-SIM) imaging showed that mitochondrial morphology in BMSCs underwent a shift toward elongated and interconnected networks over time. The transcriptional profile showed that genes associated with skeletal morphogenesis, bone development, and extracellular matrix organization were significantly upregulated in 3D culture systems. These findings indicate that 3D culture is associated with a transcriptional profile enriched in pathways commonly observed during in vivo osteogenesis, which can inform scaffold-based bone-regeneration strategies.