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.