SHED-derived conditioned exosomes enhance the osteogenic differentiation of PDLSCs via Wnt and BMP signaling in vitro.

The exosomes from human exfoliated deciduous teeth (SHED-Exos) have exhibited potential therapeutic role in dental and oral disorders. The biological effects of exosomes largely depend on cellular origin and physiological status of donor cell. In the present study, we explored the influence of conditioned exosomes from SHED with osteogenic induction on periodontal ligament stem cells (PDLSCs) in vitro. Conditioned SHED-Exos from a 3-day osteogenic supernatant were applied during PDLSCs osteogenic differentiation. We found that conditioned SHED-Exos had no cytotoxicity on PDLSCs viability assessed by CCK-8 assay. These SHED-Exos promoted PDLSCs osteogenic differentiation with deep Alizarin red staining, high alkaline phosphatase (ALP) activity and upregulated osteogenic gene expression (RUNX2, OPN and OCN). We further found BMP/Smad signaling and Wnt/β-catenin were activated by enhanced Smad1/5/8 phosphorylation and increased nuclear β-catenin protein expression. Inhibiting these two signaling pathways with specific inhibitors (cardamonin and LDN193189) remarkably weakened the enhanced osteogenic differentiation. Furthermore, Wnt3a and BMP2 were upregulated in SHED and SHED-Exos. Silencing Wnt3a and BMP2 in SHED-Exos partially counteracts the enhanced osteogenic differentiation. Our findings indicate that conditioned SHED-Exos-enhanced PDLSCs osteogenic differentiation was partly due to its carrying Wnt3a and BMP2. These data provide new insights into the use of SHED-Exos in periodontitis-induced bone defects therapy.

Wang M ，Li J ，Ye Y ，He S ，Song J ... -  《-》

PAR1 Activation, via LMBR1/BMP Axis, Promotes the Osteogenesis of Periodontal Ligament Stem Cells (PDLSCs) and Alleviates the Inhibitory Effect of Sodium Butyrate on PDLSCs Osteogenesis.

Xia L ，Yang Z ，Hu C 《-》

microRNA-132 inhibits osteogenic differentiation of periodontal ligament stem cells via GDF5 and the NF-κB signaling pathway.

Periodontal ligament stem cells (PDLSCs) could differentiate into osteoblasts and have a great prospect in treating bone diseases. microRNAs (miRs) and nuclear factor kappa-B (NF-κB) signaling pathway have proved pivotal in regulating osteogenic differentiation. This study intended to discuss the mechanism of miR-132 and NF-κB in PDLSC osteogenesis. PDLSCs were firstly cultured, induced, and identified by detecting the surface markers and observing cell morphology. Levels of osteogenic markers alkaline phosphatase (ALP), bone morphogenetic proteins 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), along with miR-132 expression were measured. The osteoblast activity and mineral deposition were detected by ALP and alizarin red S (ARS) stainings. The targeting relationship between miR-132 and growth differentiation factor 5 (GDF5) was verified. The gain-and loss-of-function was performed to discuss roles of miR-132 and GDF5 in osteogenic differentiation of PDLSCs. Besides, levels of NF-κB signaling pathway-related proteins were measured. In osteogenic differentiation of PDLSCs, levels of ALP, BMP2, Runx2 and OCN were upregulated while miR-132 was downregulated. Overexpressing miR-132 reduced levels of osteogenic markers, osteoblast activity, ALP and ARS intensity and the activation of NF-κB axis. GDF5 is a target of miR-132 and GDF5 overexpression reversed the inhibitory effects of overexpressed miR-132 on PDLSC osteogenesis. Together, miR-132 could inhibit PDLSC osteogenesis via targeting GDF5 and activating NF-κB axis. These data provide useful information for PDLSC application in periodontal therapy.

Xu Y ，Ren C ，Zhao X ，Wang W ，Zhang N ... -  《-》

Osteogenic effect of crocin in human periodontal ligament stem cells via Wnt/β-catenin signaling.

Crocin is a major class of medicinal components in saffron. This study aimed to determine whether crocin directly promotes the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in vitro and in vivo. CCK8 cell proliferation assay, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and Alizarin Red staining were performed in PDLSCs using crocin as a stimulant. DKK1 was used to selectively inhibit Wnt/β-catenin signaling, and Western blotting was performed to investigate the underlying mechanism. The PDLSCs were mixed with calcium phosphate cement and implanted into nude mice subcutaneously to study the effect of crocin on PDLSC osteogenic differentiation in vivo. The CCK-8 assay showed that crocin did not promote the proliferation of PDLSCs. Treatment with 400 μM crocin significantly promoted PDLSC mRNA levels of ALP, COL1 and OCN; RUNX2 and BMP2 protein expression; mineralized nodule formation in vitro and in vivo; and ALP activity in tissues in vivo. In addition, crocin significantly promoted the phosphorylation of β-catenin and cyclin D1. DKK1 inhibits Wnt/β-catenin activation and partially reverses crocin-mediated promotion of PDLSC osteogenic differentiation. Crocin may contribute to the regeneration of periodontal bone tissue.

Gu Y ，Bai Y 《-》

Fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated periodontal ligament stem cells by regulating the bone morphogenetic protein 2/Smad pathway.

The aim of the present study was to investigate the role of fraxinellone in periodontitis and identify its potential mechanisms. Lipopolysaccharide-induced periodontal ligament stem cells (PDLSCs) was employed to simulate the periodontitis in vitro. The levels of inflammatory factors were evaluated. After treatment with fraxinellone, alkaline phosphatase activity was determined. Additionally, calcium nodules staining was evaluated by alizarin red staining and the expression of osteogenesis differentiation-associated proteins was detected using western blot analysis. Moreover, the levels of proteins in bone morphogenetic protein 2 (BMP2)/Smad pathway were measured. Subsequently, BMP2 was silenced by transfection with small hairpin RNA to explore the underlying mechanisms of fraxinellone in lipopolysaccharide-induced PDLSCs. Lipopolysaccharide stimulation significantly upregulated the levels of inflammatory factors, which were inhibited by fraxinellone intervention. Moreover, fraxinellone notably promoted osteogenic differentiation and calcification shown by increasing levels of alkaline phosphatase, calcification and osteogenic marker proteins. Furthermore, the expression of BMP2, phosphorylated Smad1 and phosphorylated Smad5 was remarkably upregulated when fraxinellone exposure in lipopolysaccharide-induced PDLSCs. What's more, BMP2 silencing dramatically restored the effects of fraxinellone on inflammation and osteogenic differentiation of PDLSCs stimulated by lipopolysaccharide. These data demonstrated that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs by regulating the BMP2/Smad pathway, providing experimental supports for the clinical application of fraxinellone in the treatment of periodontitis.

Fu Z ，Wang X ，Li B ，Tang Y ... -  《-》