Conclusion
In the fracture microenvironment, M1 and M2 macrophages promoted BMSC osteogenesis by secreting exosomes containing LOC103691165.
Methods
Rat BMSCs and spleen macrophages were co-cultured with serum derived from the fracture microenvironment of rat tibia. BMSC osteogenesis was evaluated using Alizarin red staining and the expression of BMP-2, RUNX2, OPN, and OC mRNA. BMSC osteogenesis was evaluated after co-culture with macrophages stimulated using hypoxic conditions or colony-stimulating factor (CSF). The uptake of macrophage-derived exosomes by BMSCs was evaluated using the exosome uptake assay. High-throughput sequencing and bioinformatics analyses were performed to identify key lncRNAs in the macrophage exosomes. The effect of lncRNA expression levels on BMSC osteogenesis was also assessed using a lncRNA overexpression plasmid and siRNA technology. M1 and M2 macrophages were distinguished using flow cytometry and the key exosomal lncRNA was detected by in situ hybridization.
Purpose
To investigate the effect of macrophage exosomal long non-coding (lnc)RNAs on bone mesenchymal stem cell (BMSC) osteogenesis and the associated mechanism.
Results
In the fracture microenvironment, macrophages (stimulated using either hypoxia or CSF) significantly increased the osteogenic ability of BMSCs. We showed that BMSCs assimilated macrophage-derived vesicles and that the inhibition of exosomal secretion significantly attenuated the macrophage-mediated induction of BMSC osteogenesis. The hypoxia condition led to the up-regulation of 310 lncRNAs and the down-regulation of 575 lncRNAs in macrophage exosomes, while CSF stimulation caused the up-regulation of 557 lncRNAs and the down-regulation of 407 lncRNAs. In total, 108 lncRNAs were co-up-regulated and 326 lncRNAs were co-down-regulated under both conditions. We eventually identified LOC103691165 as a key lncRNA that promoted BMSC osteogenesis and was expressed at similar levels in both M1 and M2 macrophages.