Platelet-rich fibrin-conditioned medium promotes osteogenesis of dental pulp stem cells through TGF-β and PDGF signaling

Abstract

Introduction

Mesenchymal stem cells (MSCs), a key cell source for regenerative medicine, are conventionally cultured in human autologous serum (HAS) or fetal bovine serum (FBS). Platelet-rich fibrin-conditioned medium (PRF-CM) has emerged as a potential alternative to FBS, promoting the osteogenic differentiation of dental pulp stem cells (DPSCs) more efficiently than FBS. However, the molecular mechanisms underlying PRF-CM-induced osteogenesis in DPSCs remain unclear. Therefore, the aim of this study was to elucidate these mechanisms.

Methods

PRF-CM was prepared from peripheral blood samples collected from three healthy donors. DPSCs were derived from the dental pulp extracted from the third molars of three healthy donors. The mRNA expression patterns of DPSCs cultured in FBS or PRF-CM were compared using RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) were analyzed by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

Results

The mean blood volume collected from the three donors was 51.3 mL, yielding a mean PRF-CM volume of 168 mL. RNA-Seq identified 2258 DEGs, and KEGG pathway and GO analysis revealed that PRF-CM upregulated biological activities associated with transforming growth factor beta (TGF-β) signaling and platelet-derived growth factor (PDGF) binding compared with FBS. Western blotting analysis revealed that pSMAD2/3 and phosphor-Akt (p-Akt) were upregulated in DPSCs treated with PRF-CM, indicating that PRF-CM upregulated TGF-β and PDGF signaling pathways. The addition of SB431542 (a TGF-β inhibitor) or imatinib mesylate (a PDGF inhibitor) to osteogenic differentiation medium with PRF-CM reduced alkaline phosphatase (ALP) activity and osteogenic marker gene expression in DPSCs, respectively.

Conclusions

PRF-CM activates TGF-β and PDGF signaling pathways, thereby promoting the osteogenic differentiation of DPSCs. Moreover, the volume of PRF-CM obtained was approximately three times greater than the blood sample volume. These findings suggest that PRF-CM could serve as an effective alternative to HAS or FBS for bone regeneration therapy using MSCs.