Tumor Necrosis Factor-α Attenuates the Osteogenic Differentiation Capacity of Periodontal Ligament Stem Cells by Activating PERK Signaling.

BACKGROUND: Human periodontal ligament stem cells (PDLSCs) display efficient osteogenic differentiation capacity but fail to rescue bone breakdown associated with periodontitis. Endoplasmic reticulum (ER) stress and the unfolded protein response have recently been linked to inflammation and osteogenic differentiation. Therefore, the role of the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) pathway in the impaired osteogenic differentiation ability of PDLSCs treated with tumor necrosis factor (TNF)-α was investigated. METHODS: PDLSCs were isolated and stimulated with osteogenic media containing 1, 10, or 20 ng/mL TNF-α. Assessment included: 1) expression of runt-related transcription factor 2 and osteocalcin; 2) mRNA expression and activity of alkaline phosphatase; and 3) formation of mineralization nodules. Furthermore, expression of PERK pathway-related factors: 1) glucose-regulated protein (GRP) 78; 2) PERK; 3) activating transcription factor (ATF) 4; and 4) CCAAT-enhancer-binding proteins (C/EBP) homologous protein were also measured. Osteogenic differentiation and inhibition of the PERK pathway were also examined in cells pretreated with an inhibitor of ER stress, 4-phenylbutyric acid (PBA), followed by TNF-α stimulation. Finally, PERK small interfering RNA was used to examine osteogenic differentiation attenuated by TNF-α. RESULTS: Higher concentrations of TNF-α (10 and 20 ng/mL) impaired osteogenic differentiation of PDLSCs but activated the PERK pathway. Pretreatment of PDLSCs with lower concentrations of 4-PBA prevented the TNF-α-induced upregulation of GRP78, PERK, and ATF4 and recovered differentiation ability. Finally, PERK knockdown also restored osteogenic differentiation. CONCLUSION: TNF-α attenuates osteogenic differentiation ability of PDLSCs through activation of the PERK pathway.

[Extracellular signal-regulated kinase signaling pathway regulates the endothelial differentiation of periodontal ligament stem cells].

OBJECTIVE: To investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC). METHODS: Human PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed. RESULTS: Phosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05). CONCLUSIONS: The endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Improving the osteogenesis of rat mesenchymal stem cells by chitosan-based-microRNA nanoparticles.

MicroRNAs (miRNAs) play important roles in the osteogenic differentiation of stem cells. However, the application of miRNA in bone regeneration has been limited by its poor stability, low cellular uptake, and undesired immune response. In this study, chitosan (CS)/tripolyphosphate (TPP)/Hyaluronic Acid (HA) nanoparticles (CTH NPs) were prepared to deliver antimiR-138 to bone marrow mesenchymal stem cells (MSCs). The particle size, polydispersity index, and zeta potential of CTH NPs were related to the weight ratio of CS:TPP:HA. At optimum N/P ratio (20:1), the highest encapsulation efficiency was obtained. Both blank CTH NPs and CTH/antmiR-138 NPs exhibited no cytotoxicity to MSCs. A high transfection efficiency (nearly 70%) and significant enhancement of the osteogenesis of MSCs were observed. Above results demonstrated that CTH NPs was a potential candidate as an efficient non-viral miRNA vector to regulate the osteogenic differentiation of MSCs.