Human Dental Pulp Fibroblasts Research Articles

Divergent Proteomic Profiles and Uptake Mechanisms of Exosomes Derived from Human Dental Pulp Stem Cells, Endothelial Cells, and Fibroblasts.

Effective intercellular communication is crucial for tissue repair and regeneration, with exosomes playing a key role in mediating these processes by transferring proteins, lipids, and nucleic acids between cells. This study explored the mechanisms underlying the uptake of exosomes derived from human dental pulp stem cells (hDPSCs), human umbilical vein endothelial cells (HUVECs), and human fibroblasts (HFBs). Our findings revealed that hDPSCs exhibited the greatest capacity for exosome uptake across all three cell types. Moreover, exosomes originating from hDPSCs were also taken up in the highest amounts by all three cell types. Proteomic analysis uncovered significant differences in protein expression among exosomes from these different cell types, particularly in proteins related to endocytosis. Clathrin-dependent endocytosis emerged as the primary pathway for exosome uptake in hDPSCs and HUVECs, while HFBs appeared to use a different mechanism. Additionally, proteins such as fibronectin and tetraspanins were found to be highly expressed in hDPSC-derived exosomes, suggesting their potential involvement in exosome-cell interactions. This study offers new insights into exosome uptake mechanisms and highlights the potential of exosomes in advancing tissue engineering and regenerative medicine.

Effect of interleukin-17A on inflammatory mediator production in interleukin-1β-stimulated human dental pulp fibroblasts.

In response to pro-inflammatory cytokines such as interleukin (IL)-1β, dental pulp fibroblasts produce various inflammatory mediators, including IL-6, IL-8, CC chemokine ligand 20 (CCL20), and CXC chemokine ligand 10 (CXCL10), leading to the progression of pulpitis. IL-17/IL-17A (IL-17A) is a pro-inflammatory cytokine secreted by T helper (Th) 17 cells following their recruitment to inflamed sites; however, the roles of IL-17A during pulpitis remain unclear. The purpose of this study was to investigate the effect of IL-17A on IL-6, IL-8, CCL20 and CXCL10 production by human dental pulp fibroblasts (HDPFs) in vitro. IL-17A at a concentration of 100ng/ml induced the production of 10 times more IL-8 and 4 times more CXCL10, but not IL-6 and CCL20, compared to controls. Co-stimulation of HDPFs with IL-17A and IL-1β synergistically enhanced the production of IL-6, CCL20, IL-8 and CXCL10. IL-1β increased expression of IL-17 receptor/IL-17RA (IL-17R) on HDPFs. Moreover, the cell signal pathways of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) were more potently activated by simultaneous stimulation with IL-17A and IL-1β. These findings suggest that IL-17A participates in the progression of dental pulp inflammation through the enhanced production of inflammatory mediators in HDPFs.

Hypoxia-induced NLRP3 inflammasome activation via the HIF-1α/NF-κB signaling pathway in human dental pulp fibroblasts

BackgroundPrevious studies have reported the link between hypoxic conditions and NLRP3 inflammasome-mediated pulpal inflammation in the progression of pulpitis. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the role of HIF-1α in the regulation of NLRP3 inflammasome pathway via NF-κB signaling under hypoxic conditions with or without LPS in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis.MethodsHIF-1α plasmids or siRNAs were used to upregulate or downregulate HIF-1α in HDPFs, respectively. The effect of hypoxia with or without LPS on the NF-κB signaling and NLRP3 inflammasome pathway was analyzed by immunofluorescence staining, qRT-PCR, western blotting and ELISA.ResultsThe hypoxic conditions alone induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling in a time-dependent manner in HDPFs. The upregulation of HIF-1α further promoted hypoxia-induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. In comparison, downregulation of HIF-1α inhibited ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. Additionally, LPS plus hypoxia further promoted HIF-1α expression and NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group.ConclusionsHIF-1α served as a positive regulator of NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling in HDPFs in the sterile pulpal inflammation and caries-related pulpitis microenvironment. The finding of a novel functional HIF-1α-NF-κB-NLRP3 axis provides insight into the link between the hypoxic microenvironment and pulpal inflammation, thus supporting a promising therapeutic strategy for the control of pulpal inflammation.

Invasion of human dental pulp fibroblasts by Porphyromonas gingivalis leads to autophagy via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling pathway

ObjectivesPorphyromonas gingivalis is a pathogenic bacterium that causes periodontitis and dental pulp infection. Autophagy is a potential mechanism involved in inflammatory disease. This study established an in vitro model of P. gingivalis intracellular infection in human dental pulp fibroblasts (HDPFs) to investigate the effects of live P. gingivalis on HDPFs. MethodsMorphological and quantification techniques such as fluorescence microscopy, transmission electron microscopy (TEM), indirect immunofluorescence analysis, enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), and western blotting were used in this study. ResultsAfter cell invasion, P. gingivalis is mainly localized in the cytoplasm and lysosomes. Additionally, P. gingivalis activates autophagy in HDPFs by upregulating the expression of autophagy-related gene Beclin-1, activate autophagy-related gene12 (ATG12), and microtubule-associated protein light chain 3 (LC3). Furthermore, the invasion of P. gingivalis leads to increased phosphorylation of PI3K, Akt, and mTOR with the addition of rapamycin, whereas the addition of wortmannin decreased phosphorylation. This invasion of P. gingivalis, also causes an inflammatory response, leading to the upregulation of IL-1β, IL-6, and TNF-α. Rapamycin helps decrease levels of pro-inflammatory cytokines, but the addition of wortmannin increases them. These results show that the invasion of P. gingivalis can cause excessive inflammation and promote the autophagy of HDPFs, which is regulated by PI3K/Akt/mTOR. ConclusionsP. gingivalis escapes the immune system by inducing autophagy in the host cells, causing excessive inflammation. P. gingivalis regulates autophagy in HDPFs through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway.

Evaluation of Cytotoxicity of Allium sativum (Garlic Extract) against Human Dental Pulp Fibroblasts.

Vital pulp therapy procedures in primary dentition focuses on preservation and maintenance of pulp tissue that has been compromised due to caries, trauma, etc. Several pulp dressing materials have been used in primary teeth and some natural materials from the field of traditional medicine have also been introduced as medicaments in vital pulp therapy. The understanding of biologic and cytotoxic properties of newer materials is important for safe clinical usage. The biologic compatibility of these newer materials is imperative to limit or avoid tissue irritation or degeneration. To evaluate the cytotoxic effects of Allium sativum on cultured human primary dental pulp fibroblasts. Primary pulp fibroblasts were cultured from the pulp tissue obtained from extracted deciduous primary canines and central incisor teeth. The freshly prepared concentrations of 1000, 500, 250, 125, and 62.5 µg/mL A. sativum extract were added to the 96-well plate in triplicates to which culture medium containing fourth passage cell suspension was added previously. Cells without treatment served as control, while cells treated with 5% dimethyl sulfoxide (DMSO) served as toxic control. After the addition of experimental and control agents, cells were incubated for 24 and 48 hours at 37°C in 5% CO2 atmosphere. After the incubation period, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the number of viable cells. Absorbance was read with a microplate reader at 570 nm wavelength and the relative viability of dental pulp fibroblasts at various concentrations was expressed as color intensity of the experimental wells relative to that of control. The percentage of cell viability was also calculated accordingly. The MTT assay results revealed that A. sativum extract, in all the concentrations tested at both the time intervals maintained a cell viability of greater than 90%. At 24 hours, the mean absorbance value of untreated control wells was recorded as 0.84400 ± 0.00916 with 100% cell viability. Among all the concentrations of garlic extract tested, highest mean absorbance value of 0.83933 ± 0.00550 with 99.44% cell viability was recorded for 62.5 µg/mL concentration. At 48 hours, the mean absorbance value of untreated control wells was recorded as 1.22767 ± 0.01106 with 100% cell viability, and the highest mean absorbance value of 1.22567 ± 0.01006 with 99.83% cell viability was recorded for 62.5 µg/mL concentration. The cell viability did not seem to be affected by the concentration of A. sativum extract at 24 hours. However, at 48 hours, the sensitivity of the cells was observed to be dependent on the concentration of A. sativum with a decrease in the viability of cells noted with the increase in concentration. A. sativum extract is noncytotoxic in nature and preserves the vitality of cultured human primary dental pulp fibroblasts making it a suitable material for use in vital pulp therapy procedures of primary teeth. Devaraju R, Reddy D, Paul ST, et al. Evaluation of Cytotoxicity of Allium sativum (Garlic Extract) against Human Dental Pulp Fibroblasts. Int J Clin Pediatr Dent 2024;17(2):143-148.

"Efficacy of cytotoxic effect of green tea catechins on the human periodontal fibroblasts and human dental pulp fibroblasts -An in vitro study".

Inflammation of tooth-supporting tissue and the pulp tissue is followed by wound healing and regeneration process that involves the specific type of connective tissue cells, the fibroblasts. During periodontitis and pulpitis, the inflammation of the tissue causes damage to the fibroblasts. These fibroblasts secrete collagen proteins and maintain the structural framework; along with this the inflammatory process moves toward healing where in the specific cells such as the fibroblast cells play important roles. Green tea catechins epigallocatechin-3-gallate (EGCG) being one of the major catechins is known to have multiple beneficial effects on human fibroblasts. To assess the in vitro cytotoxicity of green tea catechins on the human periodontal ligament (PDL) fibroblasts and human dental pulp fibroblasts. Human PDL fibroblasts (hPDLFs) and human dental pulp fibroblasts were isolated from the two extracted premolar teeth that were indicated for orthodontic treatment. The fibroblasts were then seeded in 96 well tissue culture plate for cell viability study. EGCG was used at different concentration to treat the cells. After 48 h; (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay was performed to determine the cell viability. The vitality of hPDLFs and human dental pulp fibroblasts was found to be inversely proportional to EGCG concentrations. hPDLFs have shown 37% proliferation at lowest concentration of EGCG used and human dental pulp fibroblasts have shown 99% viability at lowest concentration of EGCG used.

MicroRNA-22 suppresses NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokine production by targeting the HIF-1α and NLRP3 in human dental pulp fibroblasts.

To investigate the synergetic regulatory effect of miR-22 on HIF-1α and NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1β and IL-18 in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to determine the localization of miR-22-3p, NLRP3 and HIF-1α in human dental pulp tissues (HDPTs). The miR-22 mimics and inhibitor or plasmid of NLRP3 or HIF-1α were used to upregulate or downregulate miR-22 or NLRP3 or HIF-1α in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay were conducted to confirm target association. The mRNA and protein expression of HIF-1α, NLRP3, caspase-1, IL-1β and IL-18 were determined by qRT-PCR and western blotting, respectively. The release of IL-1β and IL-18 was analysed by ELISA. The significance of the differences between the experimental and control groups was determined by one-way analysis of variance, p < .05 indicated statistical significance. A decrease in miR-22 and an increase in HIF-1α and NLRP3 in HDPTs occurred during the transformation of reversible pulpitis into irreversible pulpitis compared with that in the healthy pulp tissues (p < .05). In the normal HDPTs, miR-22-3p was extensively expressed in dental pulp cells. HIF-1α and NLRP3 were mainly expressed in the odontoblasts and vascular endothelial cells. Whereas in the inflamed HDPTs, the odontoblast layers were disrupted. HDPFs were positive for miR-22-3p, HIF-1α and NLRP3. Computational prediction via TargetScan 5.1 and luciferase reporter assays confirmed that both NLRP3 and HIF-1α were direct targets of miR-22 in HDPFs. The miR-22 inhibitor further promoted the activation of NLRP3/CASP1 inflammasome pathway induced by ATP plus LPS and hypoxia (p < .05). In contrast, the miR-22 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS and hypoxia (p < .05). MiR-22, as a synergetic negative regulator, is involved in controlling the secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3 and HIF-1α. These results provide a novel function and mechanism of miR-22-HIF-1α-NLRP3 signalling in the control of proinflammatory cytokine secretion, thus indicating a potential therapeutic strategy for future endodontic treatment.

Antibacterial activity of Brazilian red propolis and in vitro evaluation of free radical production

ObjectiveThis study aimed to evaluate the antibacterial activity of crude Brazilian red propolis (BRP) extract against anaerobic bacteria involved in primary endodontic infection. Additionally, we evaluate the cell viability and free radical production of human dental pulp fibroblasts (HDPF) in direct contact with mineral trioxide aggregate (MTA) and BRP. DesignThe Minimum Inhibitory Concentration, Minimum Bactericidal Concentration (MIC, MBC) and Minimum Inhibitory Concentration of Biofilm (MICB50) of BRP against anaerobic endodontic pathogens were determined. HDPF were exposed to BRP10 (10 μg/mL), BRP50 (50 μg/mL), MTA extract (1:1, 1:2, 1:4 e 1:8), dimethyl sulfoxide 0.5% (DMSO), and cell culture medium (DMEM). The groups were tested for cell viability (MTT assay), and free radical production (reactive oxygen species - ROS, DCFH-DA probe and nitric oxide – NO, Griess reagent). The one-way ANOVA and Tukey’s tests were employed at a significance level of 5%. ResultsMIC/MBC values of BRP performed antibacterial activity for Parvimonas micra (6.25/6.25 µg/mL), Fusobacterium nucleatum (25/25 µg/mL), Prevotella melaninogenica (50/100 µg/mL), Prevotella nigrescens (50/100 µg/mL), Prevotella intermedia (50/100 µg/mL), and Porphyromonas gingivalis (50/200 µg/mL). The MICB50 values ranged from 1.56 to 50 μg/mL. BRP and MTA stimulated cell viability, emphasizing BRP10 (p = 0.007). Furthermore, it was observed that MTA 1:1, MTA 1:2, and BRP50 slightly increased ROS (p < 0.001) and NO production (p = 0.008, p = 0.007, and p < 0.001 respectively) compared to DMEM group. ConclusionsBRP exhibits good antibacterial activity against endodontic pathogens, and both BRP and MTA promote the viability of HDPF without increasing NO and ROS production.

Clustering Human Dental Pulp Fibroblasts Spontaneously Activate NLRP3 and AIM2 Inflammasomes and Induce IL-β Secretion

Clustering Human Dental Pulp Fibroblasts Spontaneously Activate NLRP3 and AIM2 Inflammasomes and Induce IL-β Secretion

Effect of tricalcium silicate cements in gene expression of COL1A1, MAPK's, and NF-kB, and cell adhesion in primary teeth' pulp fibroblasts.

Tricalcium silicate cements (TSCs) regulate gene expression and cell responses from dental tissues surrounding the repair site. The study aimed to evaluate the gene expression levels of Collagen Type I Alpha 1 Chain (COL1A1), Mitogen-Activated Protein Kinases (MAPK's), Nuclear Factor-Kappa B (NF-κB), cell adhesion, and morphology of human dental pulp fibroblasts (hDPFs) from primary teeth treated with eluates obtained from Mineral Trioxide Aggregate (MTA) and Biodentine. hDPFs were treated with eluates from Biodentine and MTA (2.5 mg/mL in culture medium). The control group was a culture without the eluates. Gene expressions of COL1A1, MAPK's, and NF-κB were evaluated using Polymerase Chain Reaction (PCR) and cell adhesion by immunocytochemistry for Vinculin and Integrin β1 expression. Gene expression of MAPK's and NF-κB in hDPFs with the eluates from MTA and Biodentine showed no significant difference versus the control group (p > 0.05), but COL1A1 exhibited a significant difference (p < 0.05). The expression of COL1A1, MAPK's, and NF-κB was lower in cultures with MTA and Biodentine eluates regarding the control group, with no significant difference between MTA and Biodentine (p > 0.05). After 72 h of incubation, the hDPFs cultured with MTA and Biodentine eluates showed an elongated morphology; after 7 d, a loss or/and reduction of the cytoplasmic processes, and smaller nuclei were observed. Vinculin and Integrin β1 were expressed in hDPFs treated with MTA and Biodentine eluates. MTA and Biodentine did not inhibit or generate a significant difference in the expression levels of COL1A1, MAPK's, and NF-κB in hDPFs.

Effect of 4-Allyl-1-hydroxy-2-methoxybenzene (eugenol) in the expression of genes involved in cellular cycle and apoptotic process in dental pulp fibroblasts

Objective This study sought to evaluate the effect of eugenol on the cell morphology and expression of genes involved in the apoptotic process in human dental pulp fibroblasts (hDPFs) from deciduous teeth. Materials and methods hDPFs were cultured with 4 concentrations of eugenol (0.06 nM, 0.6 nM, 6 nM, 12 nM) and compared with a control group. After a 72 h incubation period, the cytotoxic effect on cell morphology by optical microscopy and gene expression by RT-PCR were evaluated. Results At 0.06 nM and 0.6 nM eugenol concentrations, vacuolisation of the cytoplasm was observed with atypical granulation of the hDPFs, and, at 6 nM and 12 nM cytoplasmic extensions disappeared almost completely. Casp-3, Casp-9, and telomerase genes were not expressed at the concentrations evaluated nor in the control group. The relative expression responses of Bcl-2 and TGF-β genes were overexpressed at the 4 concentrations. MAKP’s 0.06 nM (p < .001), 0.6 nM (p < .05) and 12 nM (p < .05) and Cyclin 1 at 12 nM showed significant difference versus the control group (p < .05). Conclusion Eugenol is capable of causing morphological changes in hDPFs in a dose-dependent manner, higher concentrations may promote overexpression of apoptotic genes.

A novel tooth bleaching gel based on peroxymonosulfate/polyphosphates advanced oxidation process: Effective whitening avoiding pulp damage and sensitivity

Pulp damage and bleaching sensitivity caused by existing hydrogen peroxide (HP) bleaching gel is a major challenge that needs to be solved urgently in the field of vital bleaching, mainly due to HP’s intrinsic high penetrability and strong oxidative property. The present study has successfully developed a novel non-hydrogen peroxide bleaching system based on peroxymonosulfate (PMS)/polyphosphates advanced oxidation process, with suitable penetration depth in dental structure. In addition to oxidizing active ingredients similar to HP gel, the new PMS gel also contains its characteristic free radical---•SO4−. Benefitting from excellent oxidative performance, novel bleaching gel exhibits a favorable tooth whitening effect. And tooth color stability after bleaching is better because it only induces a minor change on the enamel surface. Furthermore, cytological studies demonstrate that the new gel has less damage to human dental pulp fibroblasts (cell viability reaches 87.5% after 2 h’ exposure), which is mainly due to the relatively low content of oxidant (15.4%) and limited penetration depth (middle dentin). More excitingly, histological results show PMS gel applied on the molars of rats induce no inflammation of pulp tissues and no activation of pain-related receptors or peptides, consistent with the performance of blank control. These findings suggest that the new PMS/polyphosphates bleaching system has the advantages of good bleaching effectiveness, minor enamel alterations and non-damaged pulp tissue. It is expected to provide a new strategy for clinical vital bleaching treatment.

The role of fibroblasts in the modulation of dental pulp inflammation

Dental pulp fibroblasts can protect dental pulp from microbial invasion. However, little is known about the interaction between pulp fibroblasts and the immune cells. In this study, the production of proinflammatory cytokines related to inflammatory cell recruitment was evaluated in tumor necrosis factor (TNF)-α-stimulated human dental pulp fibroblasts (HDPFs). The role of TNF-α-stimulated HDPFs in the cell fusion under inflammatory process was determined with the cell co-culture with peripheral blood mononuclear cells (PBMCs). HDPFs were stimulated with various concentrations of TNF-α, and the secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 was analyzed by the enzyme-linked immunosorbent assay. The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1), macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) were determined by real-time quantitative polymerase chain reaction. TNF-α-treated HDPFs were co-cultured with PBMCs for 21 days, and characteristics of cell differentiation were assessed. TNF-α induced IL-6, IL-8 and MCP-1 production in HDPFs. Moreover, mRNA expression levels of ICAM-1, M-CSF and OPG were significantly increased in TNF-α-treated HDPFs. Co-culture of TNF-α-treated HDPFs and PBMCs stimulated formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and the F-actin rings were observed in these multinucleated cells. Our results indicate that under the stimulation of TNF-α, HDPFs may amplify inflammatory response by cytokines production, which in turn can modulate the differentiation of immune cells.

Photobiomodulation effect of red LED (630 nm) on the free radical levels produced by pulp cells under stress conditions

The aim of this study was to assess the ability of red light emitting diodes (LED) to modulate oxidative stress in human dental pulp fibroblasts (HDPFs) when different irradiation parameters are employed. Cells from primary teeth were seeded (100,000 cells/well) in 24-well plates in culture medium (DMEM). At 24 h after incubation, the culture medium was replaced with DMEM containing 10 μg/mL lipopolysaccharide (LPS). Thereafter, the cells were irradiated (LED 630 nm, 0.04 W/cm2 and 0.08 W/cm2) at 0 J/cm2 (control group), 4 J/cm2, 15 J/cm2, and 30 J/cm2; and their viability (MTT assay), number (Trypan Blue), synthesis of nitric oxide (NO) (Griess reagent), and reactive oxygen species (ROS) (fluorescence probe, DCFH-DA) were assessed. The Kruskal-Wallis and Mann-Whitney statistical tests using Bonferroni correction were employed (significance level of 5%). Compared to that in control fibroblasts, increased viability was observed in HDPFs exposed to LPS and irradiated with 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 4 J/cm2 and 15 J/cm2 at 0.08 W/cm2 (p < 0.05). Exposure to 4 J/cm2 at 0.04 W/cm2 and 15 J/cm2 and 30 J/cm2 at 0.08 W/cm2 modulated the oxidative stress in cells relative to that observed in non-irradiated LPS-treated pulp cells (p < 0.05). It was concluded that the irradiation strategies of using red LED with radiant exposures of 15 J/cm2 and 30 J/cm2 at 0.04 W/cm2 and 15 J/cm2 at 0.08 W/cm2 were the best parameters to decrease NO and ROS concentration and to stimulate viability of HDPFs exposed to LPS challenge.

PECAM1 Combines With CXCR4 to Trigger Inflammatory Cell Infiltration and Pulpitis Progression Through Activating the NF-κB Signaling Pathway.

Pulpitis is a frequent bacterially driven inflammation featured with the local accumulation of inflammatory products in human dental pulps. A GEO dataset GSE16134 comprising data of inflamed dental pulp tissues was used for bioinformatics analyses. A protein-protein interaction (PPI) analysis suggested that chemokine receptor 4 (CXCR4) owned a high correlation with platelet endothelial cell adhesion molecule-1 (PECAM1). A rat model with pulpitis was established, and lipopolysaccharide (LPS)-induced human dental pulp fibroblasts (HDPFs) were used for in vitro experiments. Then, high expression of PECAM1 and CXCR4 was validated in the inflamed dental pulp tissues in rats and in LPS-induced HDPFs. Either downregulation of PECAM1 or CXCR4 suppressed inflammatory cell infiltration in inflamed tissues as well as the inflammation and apoptosis of HDPFs. A transcription factor myocyte-enhancer factor 2 (MEF2C) was predicted and validated as a positive regulator of either PECAM1 or CXCR4, which activated the NF-κB signaling pathway and promoted pulpitis progression. To sum up, this study suggested that MEF2C transcriptionally activates PECAM1 and CXCR4 to activate the B-cell and NF-κB signaling pathways, leading to inflammatory cell infiltration and pulpitis progression.

Freeze-Dried Matrices for Buccal Administration of Propranolol in Children: Physico-Chemical and Functional Characterization.

Buccal matrices represent a widely accepted dosage form permitting a convenient, easy, reliable drug administration and reducing administration errors. The aim of this study was the development of mucoadhesive buccal matrices for propranolol administration in children. Matrices were obtained by freeze-drying of drug loaded polymeric solutions based on gum tragacanth (GT), pectin (PEC), hydroxypropylmethylcellulose (HPMC), sodium hyaluronate (HA), gelatin (GEL), chitosan (CH) or a mixture of CH and HPMC (CH/HPMC). Matrices were characterized for drug solid state, morphology, water-uptake, mucoadhesion ability, invitro drug release and permeation through porcine epithelium. The most promising formulations were tested for invitro biocompatibility in human dental pulp fibroblasts. The preparative method and the polymeric composition influenced the drug solid state, as a complete amorphization as well as different polymorphic forms were observed. GEL and PEC guaranteed a fast and complete drug release due to their rapid dissolution, while for the other matrices the release was influenced by drug diffusion through the viscous gelled matrix. Moreover, matrices based on CH and CH/HPMC showed the best mucoadhesive properties, favoured the drug permeation, in virtue of CH ability to interfere with the lipid organization of biological membrane, and were characterized by a good biocompatibility profile.

MicroRNA-223 negatively regulates LPS-induced inflammatory responses by targeting NLRP3 in human dental pulp fibroblasts.

To investigate the effect of miR-223 on NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1β and IL-18 in human dental pulp fibroblasts (HDPFs). Human dental pulp tissue (HDPT) and HDPFs were obtained from impacted third molars. The miR-223 mimics and inhibitor or NLRP3 plasmid were used to upregulate or downregulate miR-223 or NLRP3 in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay was conducted to confirm target association. The mRNA and protein expression of NLRP3, caspase-1, IL-1β and IL-18 was determined by qRT-PCR and Western blotting, respectively. The release of IL-1β and IL-18 was analysed by ELISA. The significance of the differences between the experimental and the control groups was determined using one-way analysis of variance; P<0.05 indicated statistical significance. A decrease in miR-223 and an increase in NLRP3 in HDPT occurred during the transformation of reversible pulpitis into irreversible pulpitis compared to that in healthy pulp tissue (P<0.05). The computational prediction and luciferase reporter assay confirmed that NLRP3 was a direct target of miR-223 in HDPFs. The miR-223 inhibitor further promoted ATP plus LPS-induced NLRP3/CASP1 inflammasome pathway activation compared to the ATP plus LPS-induced group (P<0.05). In contrast, the miR-223 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS compared to the ATP plus LPS-induced group (P<0.05). MiR-223 served as a negative regulator involved in the control of the production and secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3. These data provide insight into the potential regulatory effects of miRNAs on the NLRP3 inflammasome, thus opening up novel potential therapeutic avenues for future endodontic treatment.

Molecular Response of Pulp Fibroblasts after Stimulation with Pulp Capping Materials.

This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

Antibacterial activity of a glass ionomer cement doped with copper nanoparticles.

Copper nanoparticles (NCu) were synthetized and added to commercial glass ionomer cement, to evaluate in vitro its antibacterial activity against oral cavity strains. The NCu were synthesized by copper acetate reduction with L-ascorbic acid and characterized by FTIR, Raman, XPS, XRD and TEM. Then, commercial glass ionomer cement (GIC) was modified (MGIC) with various concentrations of NCu and physicochemically characterized. Cell viability was tested against human dental pulp fibroblasts (HDPFs) by Alamar-Blue assay and antibacterial test was performed against S. mutans and S. sanguinis by colony forming unit (CFU) growth method. Synthesized NCu rendered a mixture of both metallic copper and cuprous oxide (Cu2O). HDPF viability reduces with exposure time to the extracts (68-72% viability) and MGIC with 2-4 wt% NCu showed antimicrobial activity against the two tested strains.

Induction of autophagy protects human dental pulp cells from lipopolysaccharide-induced pyroptotic cell death.

The NOD-like receptor protein 3/caspase-1 inflammasome can be activated in human dental pulp tissue and fibroblasts; however, the underlying mechanisms are poorly understood. In the present study, lipopolysaccharide (LPS) was used to treat dental pulp cells to establish an inflammation model. Cell viability was examined by sulforhodamine B assay. Interleukin (IL)-1β, caspase-1, microtubule-associated protein-1 light chain 3-II/I and p62 were determined by western blotting and ELISA. The phosphorylation (p-) levels of NF-κB and NF-κB inhibitor (IκB)α protein were observed by western blotting. The results demonstrated that LPS induced pyroptotic cell death in cultured dental pulp cells, which was supported by the increased levels of IL-1β, IL-18 and caspase-1. Rapamycin and 3-methyladenine (3-MA) were used to activate and inhibit autophagy, and it was observed that LPS increased autophagy and rapamycin reduced LPS-induced dental pulp cell pyroptosis. However, 3-MA aggravated LPS-induced dental pulp cell pyroptosis. In addition, LPS inhibited the expression of IκBα, but increased the expression of p-NF-κB. Compared with the LPS group, 3-MA further inhibited the expression of IκBα but promoted the expression of p-NF-κB. However, rapamycin produced the opposite results to LPS. Under LPS treatment, the NF-κB pathway inhibitor BAY11-7082 further enhanced the inhibitory effects of rapamycin, but inhibited the promoting effects of 3-MA on the protein expression levels of IL-1β and caspase-1. The results of the present study demonstrated that there is an important crosstalk between autophagy, pyroptosis and the NF-κB pathway, and that the modulation of pyroptosis in dental pulp cells may be a promising strategy to pulpitis therapy.