Potentials of BMSCs for Regulating Osteogenic-Vascular-Neural- Lymphatic Coupling in Bone Regeneration

Methods

Human BMSCs (hBMSCs) were treated with osteogenic induction and collected from 0 to 504 h for bulk RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) were identified and Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Time-Series Transcriptomic Trend Analysis were used to comprehensively analyze the possible pathways and functions involved with these DEGs. Weighted Gene Co-expression Network Analysis (WGCNA) were constructed to identify the modules and hub genes of the process. qRT-PCR and Enzyme-linked immunosorbent assay (ELISA) were porformed to validate the expression of key genes identified by RNA-Seq.

Results We found that the gene expression tendency of hBMSCs during osteogenic differentiation can be divided into a four-stage classification: the initial adaptation stage (1-24 h), the proliferation activation stage (24-72 h), the differentiation regulation stage (72-336 h) and the remodeling stability stage (336-504 h). And 72 h was identified as the key time of the osteogenic-vascular-neural-lymphatic coupling process, with typical activation of BMP, vascular endothelial growth factor (VEGF) and PPAR signaling pathways.

Four modules and closely related hub genes such as GDF5, MGP and PAPPA2 whose expression were validated by qRT-PCR and ELISA were also identified and highlighted. Conclusions Our study revealed the time temporal trends of angiogenesis, lymphangiogenesis, and neurogenesis during BMSCs osteogenic differentiation, which not only supplemented the transcriptional regulation in bone regeneration, but also provide a theoretical basis for the design of synthetic bone substitutes.