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Potentials of BMSCs for regulating osteogenic-vascular-neural-lymphatic coupling in bone regeneration
Nan Zhang1, Yuwen Luo1, Peng Luo1
1Laboratory of Bone Tissue Engineering, Beijing Laboratory of Biomedical Materials, National Center for Orthopaedics, Beijing Research Institute of Traumatology and Orthopaedics, Beijing Jishuitan Hospital, Capital Medical University, Beijing, China.
Background:
Bone regeneration assisted by synthetic bone substitutes largely depends on the integration of the vascular, neural, and lymphatic systems in the bone. Bone marrow mesenchymal stem cells (BMSCs) are the key cells for this process. However, their role in regulating the integration has not been fully characterized.
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 associated with these DEGs. Weighted Gene Co-expression Network Analysis (WGCNA) was constructed to identify the modules and hub genes of the process. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to validate the expression of key genes identified by RNA-Seq.
Results:
Time-series analysis of the hBMSCs transcriptome suggested a dynamic expression trajectory during osteogenic differentiation, which was characterized by four functional patterns: 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). Moreover, 72 h was suggested as a potential key time point in the osteogenic-vascular-neural-lymphatic coupling process based on transcriptomic analysis, with typical activation of BMP, vascular endothelial growth factor (VEGF) and PPAR signaling pathways. Four modules and closely related hub genes such as growth differentiation factor 5 (GDF5), matrix Gla protein (MGP) and pregnancy-associated plasma protein A2 (PAPPA2), whose expressions were validated by qRT-PCR and ELISA were also identified and highlighted.
Conclusions:
Our study revealed the temporal trends of angiogenesis, lymphangiogenesis, and neurogenesis during BMSCs osteogenic differentiation, which not only supplemented the transcriptional regulation in bone regeneration, but also provided a theoretical basis for the design of synthetic bone substitutes.
