Bone regeneration using exosomes derived from hMSCs stimulated by hypoxia

Background

Mesenchymal stem cells (MSCs) secrete various kinds of soluble factors such as exosomes. Exosomes are nanoparticles and are thought to play essential roles in intercellular communication. We collected exosomes from the conditioned media from human MSCs (MSCs-Exo) and reported that MSCs-Exo enhanced bone formation. Recent studies reported that harsh environments such as a low oxygen environment stimulate cells and change the composition of their soluble factors.

Aim/Hypothesis

The aim of this study is to evaluate the bone regenerative efficacy of MSCs-Exo stimulated by hypoxia.

Materials and Methods

hMSCs were purchased from LONZA. They were cultured in Dulbecco's modi&#64257 + ed Eagle's medium with exosome-free serum at 1% O2 using the BIONIX hypoxic culture kit (Sugiyamagen, Tokyo, Japan), and then the exosomes (hypo-MSCs-Exo) were collected using an ultra-centrifuge method. They were observed using transmission electron microscopy and their particle size distribution was evaluated by Nanoparticle tracking analysis. Profiling of microRNAs contained in hypo-MSCs-Exo was performed by microarray. Rat mesenchymal stem cells (rMSCs) and human umbilical endothelial cells (HUVECs) were cultured with hypo-MSCs-Exo or MSCs-Exo for 48 hours. Then, alizarin red S staining, real-time RT-PCR analysis, and tube formation assay were performed. Rat calvarial bone defect models (5 mm in diameter) were made, and then hypo-MSCs-Exo or MSCs-Exo were applied into the defects with atelocollagen sponges. After 4 weeks, the new bone formation was evaluated by radiographical and histological analyses.

Results

hypo-MSCs-Exo or MSC-Exos were observed as round-shaped 70-200 nm nanoparticles. Profiling of microRNAs revealed that there were many kinds of miRNAs contained in hypo-MSCs-Exo and they were the different composition from those contained in MSCs-Exo. Compared with MSCs-Exo, when hypo-MSCs-Exo were applied into rMSCs, they have enhanced the osteogenic differentiation and the gene expression of Alkaline Phosphatase, Runt-related transcription factor 2, Collagen type 1 &#945 + 2. hypo-MSCs-Exo also enhanced tube formation of HUVECs. In vivo, new bone formation rate was significantly higher in the hypo-MSCs-Exo group compared with in the MSCs-Exo group.

Conclusion and Clinical Implications

A low oxygen stimulated hMSCs and changed the composition of miRNAs contained in their exosomes and hypo-MSCs-Exo enhanced osteogenesis and angiogenesis.