The pluripotent factor OCT4A enhances the self-renewal of human dental pulp stem cells by targeting lncRNA FTX in an LPS-induced inflammatory microenvironment.

BACKGROUND: Regulating the pluripotency of human dental pulp stem cells (hDPSCs) is key for the self-repair of injured dental pulp. We previously found that OCT4A promotes the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs). Recent studies have shown the interaction between OCT4A and lncRNAs in pluripotency maintenance of various stem cells. The aim of this study was to explore the underlying roles and mechanisms of OCT4A and its related lncRNAs in the proliferation and multidirectional differentiation of hDPSCs in an inflammatory microenvironment. METHODS: Human lncRNA microarrays were applied to screen out the differentially expressed lncRNAs in hDPSCs between the OCT4A-overexpressing and vector groups. Lipopolysaccharide (LPS) was used to simulate the inflammatory microenvironment. The effects of OCT4A and the lncRNA FTX on the proliferation and multidifferentiation of hDPSCs were observed by the CCK-8 assay, EdU staining, real-time PCR, western blotting, and Alizarin red and oil red O staining. Bioinformatics analysis and chromatin immunoprecipitation (ChIP) assays were performed to clarify the targeted mechanism of OCT4A on FTX. The regulation by FTX of the expression of OCT4A and its downstream pluripotent transcription factors SOX2 and c-MYC was further detected by real-time PCR and western blotting. RESULTS: The microarray results showed that 978 lncRNAs (250 of which were upregulated and 728 downregulated) were potentially differentially expressed genes (fold change ≥ 2, P < 0.05). LPS stimulation attenuated the self-renewal of hDPSCs. OCT4A enhanced the cell proliferation and multidifferentiation capacities of hDPSCs in an inflammatory microenvironment, while FTX exhibited the opposite effects. OCT4A negatively regulated FTX function by binding to specific regions on the FTX promoter, thereby inhibiting the transcription of FTX. Moreover, overexpression of FTX downregulated the expression of OCT4A, SOX2 and c-MYC, whereas knockdown of FTX facilitated their expression. CONCLUSIONS: OCT4A was found to be a crucial factor maintaining the self-renewal of hDPSCs by transcriptionally targeting FTX in an inflammatory microenvironment. Moreover, we proposed a novel function of FTX in negatively regulating the pluripotency and multilineage differentiation capacity of hDPSCs. The hierarchical organization between OCT4A and FTX expanded the understanding of the network between transcription factors and lncRNAs in fine-tuning the pluripotency/differentiation balance of adult stem cells, and provided prospective targets for optimizing dental-derived stem cell sources for regenerative endodontics.

Small extracellular vesicles derived from hypoxic preconditioned dental pulp stem cells ameliorate inflammatory osteolysis by modulating macrophage polarization and osteoclastogenesis.

Extensive macrophage inflammatory responses and osteoclast formation are predominant during inflammatory or infective osteolysis. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEV) have been shown to exert therapeutic effects on bone defects. However, cultured MSCs are typically exposed to normoxia (21% O2) in vitro, which differs largely from the oxygen concentration in vivo under hypoxic conditions. It is largely unknown whether sEV derived from dental pulp stem cells (DPSCs) cultured under hypoxic conditions (Hypo-sEV) exert better therapeutic effects on lipopolysaccharide (LPS)-induced inflammatory osteolysis than those cultured under normoxic conditions (Nor-sEV) by simultaneously inhibiting the macrophage inflammatory response and osteoclastogenesis. In this study, we show that hypoxia significantly induces the release of sEV from DPSCs. Moreover, Hypo-sEV exhibit significantly improved efficacy in promoting M2 macrophage polarization and suppressing osteoclast formation to alleviate LPS-induced inflammatory calvarial bone loss compared with Nor-sEV. Mechanistically, hypoxia preconditioning markedly alters the miRNA profiles of DPSC-sEV. MiR-210-3p is enriched in Hypo-sEV, and can simultaneously induce M2 macrophage generation and inhibit osteoclastogenesis by targeting NF-κB1 p105, which attenuates osteolysis. Our study suggests a promising potential for hypoxia-induced DPSC-sEV to treat inflammatory or infective osteolysis and identifies a novel role of miR-210-3p in concurrently hindering osteoclastogenesis and macrophage inflammatory response by inhibiting NF-kB1 expression.

A deep-water ray-based blind deconvolution for a near-surface source with a bottom-moored short-aperture vertical array.

A deep-water ray-based blind deconvolution (DW-RBD) method for estimating the channel impulse response of a near-surface source with a bottom-moored vertical array is developed. The proposed DW-RBD is an alternative when the original RBD suffers from performance degradation due to the insufficient beam resolution. The signal-processing scheme coherently utilizes the information of multipath time-delay implied in the conventional wideband beamforming output. A time-delay-related compensation term is then derived based on image theory and introduced into the original RBD to enhance multipath separation. Both simulation and experimental results demonstrate the effectiveness of the proposed method.

Impact of agitation/activation strategies on the antibiofilm potential of sodium hypochlorite/etidronate mixture in vitro.

BACKGROUND: To investigate the effect of a rotary agitation method or ultrasonically activated irrigation on the antibiofilm effect of a mixture of sodium hypochlorite (NaOCl) and etidronate (1-hydroxyethylidene-1,1-bisphosphonate, HEBP) using a dual-species biofilm model in root canal system. METHODS: Mature dual-species biofilms of Enterococcus faecalis and Streptococcus gordonii were formed in root canals of mandibular premolars. Teeth were randomly allotted (n = 12) to group 1, XP-endo Finisher (XPF); group 2, ultrasonically activated irrigation (UAI); group 3, syringe-and-needle irrigation (SNI). In all groups, canals were instrumented with a rotary instrument (XP-endo Shaper) prior to irrigant agitation/activation. A mixture containing 2.5% NaOCl and 9% HEBP was used throughout the experiment. Bacterial counts from the canal were determined using qPCR before preparation (S1), after preparation (S2), and after final irrigation agitation/activation (S3). Bacterial viability within the dentinal tubules in the coronal, middle and apical root-thirds was quantified using confocal microscopy after Live/Dead staining. The bacterial counts and viability were compared between groups using one-way ANOVA and post-hoc Tukey's tests. Paired t-test was used to compare the bacterial counts within groups. RESULTS: Instrumentation alone could significantly reduce the microbial counts in all the groups (P < 0.0001). Subsequent agitation/activation resulted in significant microbial reduction only in XPF and UAI (P < 0.05), both of which reduced significantly more microbial counts than SNI (P < 0.05). Live/Dead staining revealed that XPF and UAI showed significantly greater percentage of dead bacteria within the dentinal tubules than SNI in the coronal third (P < 0.05); UAI resulted in the significantly highest percentage of dead bacteria in the middle third (P < 0.05); while there was no significant difference between the groups in the apical third (P > 0.05). CONCLUSIONS: When using the sodium hypochlorite/etidronate mixture for irrigation, final irrigant agitation/activation with XP-endo Finisher or ultrasonic can improve disinfection of the main root canal space and the dentinal tubules in the coronal third, while ultrasonically activated irrigation appears to exhibit better disinfection within dentinal tubules in the middle third.

Comparison of the efficacy of laser-activated and ultrasonic-activated techniques for the removal of tricalcium silicate-based sealers and gutta-percha in root canal retreatment: a microtomography and scanning electron microscopy study.

BACKGROUND: Tricalcium silicate-based sealers have been usually indicated for the single-cone technique and result in more residual filling materials in root canal retreatment. Passive ultrasonic irrigation and photon-initiated photoacoustic streaming have been reported to improve the removal efficacy of root canal filling materials. However, the abilities of both techniques combined with NiTi re-instrumentation to remove residual tricalcium silicate-based sealer and gutta-percha have not been compared. The aim of this study was to evaluate the efficacy of laser-activated and ultrasonic-activated techniques in vitro for the removal of the tricalcium silicate-based sealer iRoot SP and gutta-percha after standard canal retreatment procedures with the use of nickel-titanium (NiTi) rotary instruments.

A comparative study of dentinal tubule penetration and the retreatability of EndoSequence BC Sealer HiFlow, iRoot SP, and AH Plus with different obturation techniques.

OBJECTIVES: This study aimed to evaluate dentinal tubule penetration and the retreatability of EndoSequence BC Sealer HiFlow (HiFlow), iRoot SP, and AH Plus when using the single-cone (SC) or continuous wave condensation (CWC) technique. MATERIALS AND METHODS: Sixty-five single-rooted teeth were instrumented and randomly divided into 5 groups: group 1, AH Plus/CWC; group 2, iRoot SP/CWC; group 3, iRoot SP/SC; group 4, HiFlow/CWC; and group 5, HiFlow/SC. The ability to re-establish patency during endodontic retreatment was recorded, as was the time taken to reach the working length. Dentinal tubule penetration and remaining debris after retreatment were evaluated by confocal microscopy and scanning electron microscopy. Data were analyzed by Kruskal-Wallis test and Dunn's multiple comparisons test (α = 0.05). RESULTS: The HiFlow/CWC and iRoot SP/CWC groups required more time to reach the working length than groups that underwent the SC technique regardless of the sealer used (P < .05). The HiFlow/CWC group showed a significantly higher percentage of sealer penetration area than that of the iRoot SP/SC at 4 mm from the apex (P < .05) and penetrated deeper into dentinal tubules than iRoot SP/SC at both 8-mm and 12-mm levels (P < .05). Moreover, the HiFlow/CWC and HiFlow/SC groups demonstrated less remaining sealer along the canal wall than AH Plus/CWC group at 4-mm level (P < .05). CONCLUSIONS: HiFlow/CWC technique showed better performance in dentinal tubule penetration than that of iRoot SP/SC. Both HiFlow and iRoot SP combined with CWC technique groups required more retreatment time than the other groups. Furthermore, using HiFlow with either the CWC or SC technique left less remaining sealer at 4-mm level than using AH Plus with the CWC technique during retreatment. CLINICAL RELEVANCE: With favorable performance in dentinal tubule penetration and retreatability in endodontic retreatment, the combined use of EndoSequence BC Sealer HiFlow with the recommended continuous wave condensation technique may be a worthwhile choice in root canal treatment.

Use of multipath time-delay ratio for source depth estimation with a vertical line array in deep water.

In this paper, a method for the problem of depth estimation of a broadband source via reliable acoustic path propagation is presented for the case using a vertical line array (VLA). The estimates are determined by two kinds of multipath time-delay ratios, namely, the ratio of direct-surface-reflected (D-SR) to direct-direct time-delays and the ratio of D-SR to surface-reflected-surface-reflected time-delays. The innovation of ratio behavior is that it provides a mechanism for obtaining a useful depth interval with the assumption of plane-wave propagation. The estimation accuracy of a depth interval relies on the degree to which the actual acoustic propagation characteristic can be modeled by image theory. Furthermore, the variability of depth interval due to the approximation made in the derivation method allows one to achieve binary discrimination of both the source depth and source range with only a minimal amount of prior environmental knowledge. The methodology of multipath time-delay estimation is first reviewed and improved, followed by an illustration of the source depth estimation and a discussion of the performance analysis using results from numerical simulations. Finally, the proposed method is demonstrated with experimental data collected in the South China Sea in which a short-aperture VLA is deployed near the sea bottom.

Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells.

Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.

Enterococcus faecalis immunoregulates osteoclastogenesis of macrophages.

Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.

Luteolin and apigenin activate the Oct-4/Sox2 signal via NFATc1 in human periodontal ligament cells.

Identifying small molecules to activate the Oct-4/Sox2-derived pluripotency network represents a hopeful and safe method to pluripotency without genetic manipulation. Luteolin and apigenin, two major bioactive flavonoids, enhance reprogramming efficiency and increase expression of Oct-4/Sox2/c-Myc, albeit the detailed mechanism regulating pluripotency in dental-derived cells remains unknown. In the present study, to elucidate the effect of luteolin/apigenin on pluripotency of periodontal ligament cells (PDLCs) through interaction with downstream signals, we examined cell cycle, proliferation, apoptosis, expression of Oct-4/Sox2/c-Myc, and multilineage differentiation of PDLCs with luteolin/apigenin treatment. Moreover, we profiled the differentially expressed pluripotency genes by PCR arrays. Our results demonstrated that luteolin/apigenin restrained cell proliferation, increased apoptosis, and arrested PDLCs in G2/M and S phase. Luteolin and apigenin activated expression of Oct-4, Sox2, and c-Myc in a time- and dose-dependent pattern, and repressed lineage-specific differentiation. PCR arrays profiled multiple signals in PDLCs with luteolin/apigenin treatment, among which NFATc1 was the major upregulated gene. Notably, blocking of the NFATc1 signal with INCA-6 significantly decreased mRNA and protein expression of Oct-4, Sox2, and c-Myc in PDLCs with luteolin/apigenin treatment, indicating that NFATc1 may act as an upstream modulator of Oct-4/Sox2 signal. Taken together, this study showed that luteolin and apigenin effectively maintain pluripotency of PDLCs through activation of Oct-4/Sox2 signal via NFATc1.

XPC Promotes Pluripotency of Human Dental Pulp Cells through Regulation of Oct-4/Sox2/c-Myc.

Introduction. Xeroderma pigmentosum group C (XPC), essential component of multisubunit stem cell coactivator complex (SCC), functions as the critical factor modulating pluripotency and genome integrity through interaction with Oct-4/Sox2. However, its specific role in regulating pluripotency and multilineage differentiation of human dental pulp cells (DPCs) remains unknown. Methods. To elucidate the functional role XPC played in pluripotency and multilineage differentiation of DPCs, expressions of XPC in DPCs with long-term culture were examined by real-time PCR and western blot. DPCs were transfected with lentiviral-mediated human XPC gene; then transfection rate was investigated by real-time PCR and western blot. Cell cycle, apoptosis, proliferation, senescence, multilineage differentiation, and expression of Oct-4/Sox2/c-Myc in transfected DPCs were examined. Results. XPC, Oct-4, Sox2, and c-Myc were downregulated at P7 compared with P3 in DPCs with long-term culture. XPC genes were upregulated in DPCs at P2 after transfection and maintained high expression level at P3 and P7. Cell proliferation, PI value, and telomerase activity were enhanced, whereas apoptosis was suppressed in transfected DPCs. Oct-4/Sox2/c-Myc were significantly upregulated, and multilineage differentiation in DPCs with XPC overexpression was enhanced after transfection. Conclusions. XPC plays an essential role in the modulation of pluripotency and multilineage differentiation of DPCs through regulation of Oct-4/Sox2/c-Myc.