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Abstract Background Pulp inflammation is complex interactions between different types of cells and cytokines. To mimic the interactions of different types of cells in inflamed dental pulp tissues, dental pulp cells (DPCs) were cocultured with different ratios of macrophages (THP-1) or LPS treatment. Methods DPCs were cocultured with various ratios of THP-1, then photographed cell morphology and determined cell viability by MTT assay at preset times. Total RNA was also extracted to measure the inflammation marker-IL-6 and IL-8 expressions by RT-Q-PCR. The DPCs and THP-1 were treated with 0.01 – 1μg/ml lipopolysaccharide (LPS) and extract RNA at preset times, and detected IL-6 and IL-8 expression. DPCs were cocultured with various ratios of THP-1 with 0.1 μg/mL LPS, and detected IL-6 and IL-8 expression after 24 and 48 h. The data were analyzed by unpaired t-test or Mann-Whitney test. Differences were considered statistically significant when p

Background: Pulp inflammation is complex interactions between different types of cells and cytokines. To mimic the interactions of different types of cells in inflamed dental pulp tissues, dental pulp cells (DPCs) were cocultured with different ratios of macrophages (THP-1) or LPS treatment. Methods: DPCs were cocultured with various ratios of THP-1, then photographed cell morphology and determined cell viability by MTT assay at preset times. Total RNA was also extracted to measure the inflammation marker-IL-6 and IL-8 expressions by RT-Q-PCR. The DPCs and THP-1 were treated with 0.01 – 1μg/ml lipopolysaccharide (LPS) and extract RNA at preset times, and detected IL-6 and IL-8 expression. DPCs were cocultured with various ratios of THP-1 with 0.1 μg/mL LPS, and detected IL-6 and IL-8 expression after 24 and 48 h. The data were analyzed by unpaired t-test or Mann-Whitney test. Differences were considered statistically significant when p < 0.05. Results: THP-1 and DPCs coculture models did not suppress the viability of DPCs and THP-1. Cocultured with various ratios of THP-1 could increase IL-6 and IL-8 expressions of DPCs (p = 0.0056 - p < 0.0001). The expressions of IL-6 and IL-8 were stronger in higher ratio groups (p = 0.0062 - p < 0.0001). LPS treatment also induced IL-6 and IL-8 expressions of DPCs and THP-1 (p = 0.0179 – p < 0.0001 and p = 0.0189 – p < 0.0001, separately). Under the presence of 0.1 μg/mL LPS, DPCs cocultured with THP-1 for 24 h also enhanced IL-6 and IL-8 expression (p = 0.0022). After cocultured with a higher ratio of THP-1 for 48 h, IL-6 and IL-8 expressions were even stronger in the presence of LPS (p = 0.0260). Conclusions: Coculturing dental pulp cells and macrophages under LPS treatment aggravate the inflammatory process. The responses of our models were more severe than traditional inflamed dental models and better represented what happened in the real dental pulp. Utilizing our models to explore the repair and regeneration in endodontics will be future goals. [ABSTRACT FROM AUTHOR]

Objectives: To explore the inflammatory and differentiation response in inflamed dental pulp cells (DPCs) induced by lipopolysaccharide (LPS) under different conditions with Biodentine and mineral trioxide aggregate (MTA) treatment. Materials and methods: DPCs were treated with 0.001–1 µg/mL LPS for different periods to induce inflammation. Normal and inflamed DPCs were further treated with 0.14 mg/mL Biodentine or 0.13 mg/mL MTA for different periods. mRNA expression level of IL-6, IL-8 and ALP were analysed by qPCR. DSPP protein expression was detected by western blot. The data were analysed by the Mann–Whitney test, unpaired t test or two-way ANOVA. Results: After treatment for different times and with different concentrations of LPS, different severity of pulp inflammation was revealed by the expressions of IL-6 and IL-8. Higher concentrations of LPS induced higher IL-6 and IL-8 expressions, and these expressions first increased and then decreased (p < 0.0001). At 96 and 192 h, Biodentine significantly suppressed IL-6 expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine suppressed ALP expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine induced DSPP expressions in both normal and inflamed DPCs (p < 0.05). Conclusion: Biodentine enhanced more DSPP differentiation of both normal and inflamed DPCs under different treatment durations than MTA. Clinical relevance: The prognosis of vital pulp therapy may depend on the severity of pulp inflammation which is difficult to be determined in clinical settings. Therefore, Biodentine may enhance odontogenic differentiation in different severity of pulp inflammation imply its clinical indications. [ABSTRACT FROM AUTHOR]

Objective The aim of this study is to investigate the roles of Notch signaling and autophagy on mineral trioxide aggregate (MTA) induced differentiation of human dental pulp cells (hDPCs). Methods Third molars from healthy human were collected and hDPCs were isolated by a combined digestion of collagenase Ⅰ and dispaseⅡ. Real time PCR were used to test the mRNA expression levels of alkaline phosphatase (ALP), runt⁃related transcription factor 2 (Runx2) and dentin sialophoprotein (DSPP) in MTA treated hDPCs in different time (24 h, 3 d and 7 d). The mineral⁃ ization nodules formed by hDPCs with or without MTA treatment were detected by Von Kossa staining. Expressions of Notch1, Jagged1, Hes1, LC3Ⅱ/LC3 Ⅰand p62 in wild type and MTA treated hDPCs were detected by western blotting. Results MTA extracted in a concentration of 0.1 mg/mL could promote the differentiation of hDPCs. Compared with that of wild type hDPCs, the expressions of Notch1, Hes1, or Jagged1 and p62 (P＜0.01) in MTA treated hDPCs were significantly increased. MTA treatment showed inhibition effects on autophagy flux similar to Bafilomycin A1, a specific inhibitor of fusion between autophagosomes and lysosomes. Conclusion MTA could promote hDPCs differentiation with highly relevant in stimulating Notch1⁃Jagged1⁃Hes1 signaling and inhibition of autophagy flux.

Multicellular three‐dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two‐dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia‐inducible factor 1‐α (HIF‐1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF‐1α and VEGF. The treatment with YC‐1 (inhibitor to HIF‐1α) blocked the upregulation of VEGF, indicating a downstream event to HIF‐1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y‐27632 (inhibitor to Rho‐associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho‐associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF‐1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC‐based research and application. Our results demonstrate that chitosan and poly(vinyl alcohol) membranes support human dental pulp progenitor cells aggregation to form multicellular spheroids. Confocal immunofluorescence images show the expression of HIF‐1α in the cells throughout the spheroids. The 3D spheroids formation on chitosan membrane and subsequently enhanced cell migration ability are involved with RhoA‐mediated signaling pathway. [ABSTRACT FROM AUTHOR]

In this study, we investigated the anti-inflammatory, odontogenic and pro-angiogenic effects of integrating simvastatin and nanofibrous poly( l -lactic acid) (NF-PLLA) scaffolds on dental pulp cells (DPCs). Highly porous NF-PLLA scaffolds that mimic the nanofibrous architecture of extracellular matrix were first fabricated, then seeded with human DPCs and cultured with 0.1 μM simvastatin and/or 10 μg/mL pro-inflammatory stimulator lipopolysaccharide (LPS). The gene expression of pro-inflammatory mediators (TNF-α, IL-1β and MMP-9 mRNA) and odontoblastic markers (ALP activity, calcium content, DSPP, DMP-1 and BMP-2 mRNA) were quantified after long-term culture in vitro . In addition, we evaluated the scaffold’s pro-angiogenic potential after 24 h of in vitro co-culture with endothelial cells. Finally, we assessed the combined effects of simvastatin and NF-PLLA scaffolds in vivo using a subcutaneous implantation mouse model. The in vitro studies demonstrated that, compared with the DPC/NF-PLLA scaffold constructs cultured only with pro-inflammatory stimulator LPS, adding simvastatin significantly repress the expression of pro-inflammatory mediators. Treating LPS+ DPC/NF-PLLA constructs with simvastatin also reverted the negative effects of LPS on expression of odontoblastic markers in vitro and in vivo . Western blot analysis demonstrated that these effects were related to a reduction in NFkBp65 phosphorylation and up-regulation of PPARγ expression, as well as to increased phosphorylation of pERK1/2 and pSmad1, mediated by simvastatin on LPS-stimulated DPCs. The DPC/NF-PLLA constructs treated with LPS/simvastatin also led to an increase in vessel-like structures, correlated with increased VEGF expression in both DPSCs and endothelial cells. Therefore, the combination of low dosage simvastatin and NF-PLLA scaffolds appears to be a promising strategy for dentin regeneration with inflamed dental pulp tissue, by minimizing the inflammatory reaction and increasing the regenerative potential of resident stem cells. Statement of Significance The regeneration potential of stem cells is dependent on their microenvironment. In this study, we investigated the effect of the microenvironment of dental pulp stem cells (DPSCs), including 3D structure of a macroporous and nanofibrous scaffold, the inflammatory stimulus lipopolysaccharide (LPS) and a biological molecule simvastatin, on their regenerative potential of mineralized dentin tissue. The results demonstrated that LPS upregulated inflammatory mediators and suppressed the odontogenic potential of DPSCs. Known as a lipid-lowing agent, simvastatin was excitingly found to repress the expression of pro-inflammatory mediators, up-regulate odontoblastic markers, and exert a pro-angiogenic effect on endothelial cells, resulting in enhanced vascularization and mineralized dentin tissue regeneration in a biomimetic 3D tissue engineering scaffold. This novel finding is significant for the fields of stem cells, inflammation and dental tissue regeneration. [ABSTRACT FROM AUTHOR]

We have isolated dental pulp cells (DPCs) from three healthy (hDPCs) and three carious (cDPCs) donors and shown that compared to hDPCs cells isolated from superficial carious lesions show higher clonogenic potential; show an equivalent proportion of cells with putative stem cell surface markers; show enhanced matrix mineralization capability; have enhanced angiogenic marker expression and retain the inflammatory phenotype in vitro characteristic of superficial caries lesions in vivo. Our findings suggest that cDPCs may be used for further investigation of the cross talk between inflammatory, angiogenic and mineralization pathways in repair of carious pulp. In addition cells derived from carious pulps (almost always discarded) may have potential for future applications in mineralized tissue repair and regeneration.

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. [ABSTRACT FROM AUTHOR]

Objective: Mineral trioxide aggregate (MTA), a wildly used pulp capping material, could affect the proliferation and differentiation of dental pulp cells.The aim of this study is to study the roles of Notch signalling and autophagy in MTA induced human dental pulp cells (hDPCs) proliferation promotions. Methods: Healthy human third molars were collected and hDPCs were isolated by a combined digestion of collagenase Ⅰ and dispase Ⅱ.MTA extracts of different concentrations (0.5, 1.0, 2.0, 5.0, 10.0 mg/mL) were used to test the cytotoxicity by cells counting kit (CCK-8) assays and to select the optimum concentration for hDPCs survival.. Expressions of Notch1, Hes1, LC3Ⅱ / Ⅰ and p62 in wild type and MTA treated hDPCs were detected by western blotting. Results: MTA extracts in a concentration of 1.0 mg/mL exerted most profoundly promotion effects on the proliferation of hDPCs among all concentrations tested. MTA of high concentration (10 mg/mL) was toxically to cells.Compared with that of wild type hDPCs, the expressions of Notch1 and Hes1(P＜0.05), or p62 and LC3Ⅱ/Ⅰ (P＜0.01) in MTA treated hDPCs were significantly increased. Much lower expression of Notch1 was detected in hDPCs when autophagy was induced by Earle's balanced salt solution (EBSS) starvation for 24 h. Conclusions: MTA could up-regulated hDPCs proliferation with highly relevant in stimulating Notch1-Hes1 signalling and inhibition of autophagy. The study is supposed to provide new insight in unrevealing the mechanisms of MTA mediated dental pulp cells proliferation. [ABSTRACT FROM AUTHOR]

We have isolated dental pulp cells (DPCs) from three healthy (hDPCs) and three carious (cDPCs) donors and shown that compared to hDPCs cells isolated from superficial carious lesions show higher clonogenic potential; show an equivalent proportion of cells with putative stem cell surface markers; show enhanced matrix mineralization capability; have enhanced angiogenic marker expression and retain the inflammatory phenotype in vitro characteristic of superficial caries lesions in vivo. Our findings suggest that cDPCs may be used for further investigation of the cross talk between inflammatory, angiogenic and mineralization pathways in repair of carious pulp. In addition cells derived from carious pulps (almost always discarded) may have potential for future applications in mineralized tissue repair and regeneration. [ABSTRACT FROM AUTHOR]

Abstract: Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions. [Copyright &y& Elsevier]

Background: Intercellular communication in the environments of mature or aged cells can restore and regenerate their function and promote the expression of pluripotency markers. The regeneration of dental tissue is stimulated by periodontal ligament cells (PDLCs) and dental pulp cells (DPCs). However, the communication networks between the cells and their microenvironments are poorly understood., Methods: In this study, gene expression was analyzed by polymerase chain reaction, and chromatin immunoprecipitation assays, dual-luciferase assays, and electrophoretic mobility shift assays were used to analyze the signaling pathways associated with pluripotency after the knockdown or overexpression of caudal-type homeobox transcription factor 2 (CDX2)., Results: Elevated levels of SRY-box transcription factor 2 (Sox2) and octamer-binding transcription factor 4 (Oct-4) were observed in the co-culture system, while the levels of CDX2 were significantly reduced. The overexpression of CDX2 promoted cell apoptosis and reduced the synthesis stage of the cell cycle. CDX2 was shown to bind directly to the promoter regions of Sox2 and Oct-4. The silencing of CDX2 promoted calcium deposition, adipogenic differentiation, and elevated alkaline phosphatase (ALP) activity in the DPCs., Conclusions: These findings demonstrate the enhancement of DPC and PDLC pluripotency by intercellular communication. CDX2 plays a significant part in the regulation of DPC and PDLC pluripotency via its regulation of Oct-4 and Sox2 expression., Competing Interests: Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-3492/coif). The authors have no conflicts of interest to declare., (2022 Annals of Translational Medicine. All rights reserved.)

In this study, we investigated the anti-inflammatory, odontogenic and pro-angiogenic effects of integrating simvastatin and nanofibrous poly(l-lactic acid) (NF-PLLA) scaffolds on dental pulp cells (DPCs). Highly porous NF-PLLA scaffolds that mimic the nanofibrous architecture of extracellular matrix were first fabricated, then seeded with human DPCs and cultured with 0.1 μM simvastatin and/or 10 μg/mL pro-inflammatory stimulator lipopolysaccharide (LPS). The gene expression of pro-inflammatory mediators (TNF-α, IL-1β and MMP-9 mRNA) and odontoblastic markers (ALP activity, calcium content, DSPP, DMP-1 and BMP-2 mRNA) were quantified after long-term culture in vitro. In addition, we evaluated the scaffold's pro-angiogenic potential after 24 h of in vitro co-culture with endothelial cells. Finally, we assessed the combined effects of simvastatin and NF-PLLA scaffolds in vivo using a subcutaneous implantation mouse model. The in vitro studies demonstrated that, compared with the DPC/NF-PLLA scaffold constructs cultured only with pro-inflammatory stimulator LPS, adding simvastatin significantly repress the expression of pro-inflammatory mediators. Treating LPS+ DPC/NF-PLLA constructs with simvastatin also reverted the negative effects of LPS on expression of odontoblastic markers in vitro and in vivo. Western blot analysis demonstrated that these effects were related to a reduction in NFkBp65 phosphorylation and up-regulation of PPARγ expression, as well as to increased phosphorylation of pERK1/2 and pSmad1, mediated by simvastatin on LPS-stimulated DPCs. The DPC/NF-PLLA constructs treated with LPS/simvastatin also led to an increase in vessel-like structures, correlated with increased VEGF expression in both DPSCs and endothelial cells. Therefore, the combination of low dosage simvastatin and NF-PLLA scaffolds appears to be a promising strategy for dentin regeneration with inflamed dental pulp tissue, by minimizing the inflammatory reaction and increasing the regenerative potential of resident stem cells., Statement of Significance: The regeneration potential of stem cells is dependent on their microenvironment. In this study, we investigated the effect of the microenvironment of dental pulp stem cells (DPSCs), including 3D structure of a macroporous and nanofibrous scaffold, the inflammatory stimulus lipopolysaccharide (LPS) and a biological molecule simvastatin, on their regenerative potential of mineralized dentin tissue. The results demonstrated that LPS upregulated inflammatory mediators and suppressed the odontogenic potential of DPSCs. Known as a lipid-lowing agent, simvastatin was excitingly found to repress the expression of pro-inflammatory mediators, up-regulate odontoblastic markers, and exert a pro-angiogenic effect on endothelial cells, resulting in enhanced vascularization and mineralized dentin tissue regeneration in a biomimetic 3D tissue engineering scaffold. This novel finding is significant for the fields of stem cells, inflammation and dental tissue regeneration., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)