Effects of exosomes derived from platelet-rich plasma on osteogenic differentiation of dental pulp stem cells.

Platelet-rich plasma (PRP) can cause osteogenic differentiation of dental pulp stem cells (DPSCs). However, the effect of exosomes derived from PRP (PRP-Exos) on osteogenic differentiation of DPSCs remains unclear. Herein, we evaluated the impact of PRP-Exos on osteogenic differentiation of DPSCs. PRP-Exos were isolated and identified by transmission electron microscopy (TEM) and western blotting (WB). Immunofluorescence staining was performed to evaluate endocytosis of PRP-Exos by DPSCs. Alkaline phosphatase staining, alizarin red staining, western blot and qRT-PCR were carried out to evaluate the DPSCs osteogenic differentiation. The sequencing microRNA (miRNA) was conducted to determine the microRNA profile of PRP-Exos treated and untreated DPSCs. The results showed that endocytosis of PRP-Exos stimulated DPSCs odontogenic differentiation by elevated expression of ALP, DMP-1, OCN, and RUNX2. ALP activity and calcified nodules formation of PRP-Exos treated DPSCs were considerably elevated relative to that of the control group. MicroRNA sequencing revealed that 112 microRNAs considerably varied in PRP-Exos treated DPSCs, of which 84 were elevated and 28 were reduced. Pathway analysis suggested that genes targeted by differentially expressed (DE) miRNAs were contributed to many signaling cascades, such as the Wnt cascade. 65 genes targeted by 30 DE miRNA were contributed to Wnt signaling. Thus, it can be infered that PRP-Exos could enhance osteogenic differentiation and alter the miRNA expression profile of DPSCs.

Hsa_Circ_0005044 Promotes Osteo/Odontogenic Differentiation of Dental Pulp Stem Cell Via Modulating miR-296-3p/FOSL1.

Circular RNAs (circRNAs) are a form of RNAs that lack coding potential. The role of such circRNAs in dental pulp stem cell (DPSC) osteo/odontogenic differentiation remains to be determined. In this study, circRNA expression profiles in DPSC osteo/odontogenic differentiation process were analyzed by RNA-seq. qRT-PCR was used to confirm the differential expression of circ_0005044, miR-296-3p, and FOSL1 in DPSC osteogenic differentiation process. Circ_0005044, miR-296-3p, and FOSL1 were knocked down or overexpressed. Osteoblastic activity and associated mineral activity were monitored via alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Interactions between miR-296-3p, circ_0005044, and FOSL1 were assessed through luciferase reporter assays. Finally, an in vivo system was used to confirm the relevance of circ_0005044 to osteoblastic differentiation. As results, we detected significant circ_0005044 and FOSL1 upregulation in DPSC osteo/odontogenic differentiation process, as well as concomitant miR-296-3p downregulation. When knocking down circ_0005044 or overexpressed miR-296-3p, this significantly inhibited osteogenesis. Luciferase reporter assay confirmed that miR-296-3p was capable of binding to conserved sequences in the wild-type forms of both the circ_0005044 and FOSL1. Furthermore, knocking down circ_0005044 in vivo significantly attenuated bone formation. Therefore, the circ_0005044/miR-2964-3p/FOSL1 axis regulates DPSC osteo/odontogenic differentiation, which may provide potential molecular targets for dental-pulp complex regeneration.