INTRODUCTION

The roles of exosomes in osteoblast differentiation has been widely investigated. Low exosome production from donor cells constitutes the greatest challenges in exosome-based therapies. Spexin (SPX) is a neuropeptide that is involved in various biological activities including osteogenic differentiation and bone regeneration. Therefore, the purpose of this study was to investigate the effects of SPX on exosome production in osteogenic medium (OM)-treated MC3T3-E1 cells and SPX induced MC3T3-E1 cell-derived exosomes (OM + SPX-Exos) on osteoblast differentiation.

MATERIALS AND METHODS

To evaluate exosome yield, MC3T3-E1 cells were treated with SPX. Exosome marker expression and particle number were validated via reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and nanoparticle tracking analysis (NTA), respectively. MC3T3-E1 cells were then treated with various concentrations of OM + SPX-Exos and osteogenic medium treated MC3T3-E1 derived exosomes (OM-Exos). Cell proliferation, osteogenic differentiation marker expression, alkaline phosphatase (ALP) activity, and mineralization were evaluated using the CCK-8 assay, RT-qPCR, ALP staining, and alizarin red S staining, respectively.

RESULTS

SPX significantly increased exosome production and the expression of the exosome markers; Cd63, Rab27a and Alix in MC3T3E1 cells. Furthermore, OM + SPX-Exos significantly increased in the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase, biomineralized associated (Alpl), collagen type I alpha 1 (Col1a1), secreted phosphoprotein 1 (Spp1) and Integrin-binding sialoprotein (Ibsp) at a concentration of 5 µg/ml. ALP staining and alizarin red S staining also revealed that OM + SPX-Exos (5 µg/ml) resulted in more ALP-positive cells and markedly promoted mineralization, respectively.

CONCLUSION

In general, these results indicate that SPX stimulates exosome production. OM + SPX-Exos enhances MC3T3-E1 cells proliferation, osteogenic differentiation and mineralization.