Extracellular vesicles from the inflammatory microenvironment regulate the osteogenic and odontogenic differentiation of periodontal ligament stem cells by miR-758-5p/LMBR1/BMP2/4 axis

Abstract

Background Extracellular vesicles (EVs) play a key role in constructing a microenvironment that favors the differentiation of stem cells. The present work aimed to determine the molecular mechanisms by which EV derived from inflammatory dental pulp stem cell (iDPSC-EV) influence periodontal ligament stem cells (PDLSCs) and provide a potential strategy for bone and dental pulp regeneration. Methods The osteogenic and odontogenic differentiation was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S (ARS) staining, and immunofluorescence staining. To detect proliferation, the Cell Counting Kit-8 (CCK-8) assay, and flow cytometry analysis were used. EVs were isolated by the Exoperfect kit and ultrafiltration and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. The expression profile of miRNAs in EVs was studied using miRNA sequence and bioinformatics, and one of the upregulated miRNAs was evaluated on PDLSCs. Results The inflammatory microenvironment stimulated osteogenic and odontogenic differentiation of DPSCs and iDPSC-EV behaved alike on PDLSCs. MiR-758-5p was upregulated in iDPSC-EV and was demonstrated to play a significant role in the osteogenic and odontogenic commitment of PDLSCs. A dual-luciferase reporter assay confirmed the binding site between miR-758-5p and limb development membrane protein 1 (LMBR1). The knockdown of LMBR1 also enhanced the above potential. Mechanically, bone morphogenetic protein (BMP) signaling was activated. Conclusions EVs from the inflammatory microenvironment enhanced the osteogenic and odontogenic differentiation of PDLSCs partly by shuttering LMBR1-targeting miR-758-5p via BMP signaling.

Hsa‐miR‐27b‐5p suppresses the osteogenic and odontogenic differentiation of stem cells from human exfoliated deciduous teeth via targeting BMPR1A: An ex vivo study

“…SHEDs separated from healthy retained deciduous teeth (from children aged 6–8). The crown pulp tissues were isolated aseptically within 1 h, and the cells were incubated as mentioned previously (Yan et al, 2022). Briefly, the cells were incubated in an ɑ‐modified Eagle's minimum essential medium (α‐MEM; 12 561‐056; Gibco) with 10% fetal bovine serum (FBS; SH30070.03; Hyclone), 100 units/mL penicillin and 100 mg/mL streptomycin (10 378 016; Gibco) at 37°C in a 5% CO 2 incubator.…”

“…The total RNA of SHEDs was extracted with TRIzol reagent (10296010CN; Invitrogen) and was reverse transcripted to cDNA by PrimeScript RT Master Mix kit (R222 and R101; Vazyme). QRT-PCR was conducted using ChamQTM SYBR Green quantitative PCR Master Mix (Q712; Vazyme) and the parameters of the procedure were set according to previous study's protocol (Yan et al, 2022). Primers in this research were listed in Table S1, among which GAPDH and U6 were used as internal references for mRNAs and miR-27b-5p.…”

“…Hsa‐miR‐27b‐5p is considered to be a tumour suppressor (Ke et al, 2022; Liu et al, 2020) and can exert anti‐apoptotic roles (Pan et al, 2020). Surprisingly, our previous work certificated that miR‐27b‐5p was abundantly expressed in extracellular vehicles (EVs) originating from inflammatory‐induced dental pulp stem cells (DPSCs) (Yan et al, 2022). We speculate that miR‐27b‐5p potentially has significant regulatory effects on the differentiation of SHEDs.…”

The Applications and Potentials of Extracellular Vesicles from Different Cell Sources in Periodontal Regeneration

“…Increasing evidence suggests that SCEVs from mild inflammatory cytokine pre-stimulated cells are beneficial to periodontal bone formation. For example, CEVs derived from DPSCs pretreated with a low concentration of TNF-α (TNF-α-DPSC-EVs) delivered miR-758-5p targeting LMBR1 to activate BMP2/4 and promote the osteogenic differentiation of PDLSCs [ 69 ]. Similarly, LPS-pretreated dental follicle (stem) cell (DFC/DFSC)-derived CEVs (LPS-DFC-EVs/LPS-DFSC-EVs) promoted the migration, proliferation and osteogenic differentiation of PDLSCs, and exerted an antioxidant function through ROS/MAPK signaling pathway.…”

The role of extracellular vesicles in periodontitis: pathogenesis, diagnosis, and therapy

“…In the inflammatory microenvironment, dental pulp stem cells (DPSCs)-EVs may shutter LMBR1-targeting miR-758-5p via the BMP signaling pathway to promote osteogenic and odontogenic differentiation of PDLSCs and provide a potential strategy for bone regeneration ( 206 ). DPSCs exosomes can effectively reduce periodontal bone loss by stimulating the migration of human DPSCs and mouse osteoblasts ( 207 ).…”