Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

The Effect of Glycyrrhizin on the Viability and Proliferation of Dental Pulp Stem Cells Compared to Intracanal Medicaments.

AIM: To study the effect of glycyrrhizin (GA) on the viability and proliferation of dental pulp stem cells (DPSCs) compared with intracanal medicaments. MATERIALS AND METHODS: Third molars of an adult donor were used to obtain the DPSCs. Flow cytometry was utilized to conduct phenotypic analysis for DPSCs. The methyl-thiazol tetrazolium (MTT) test was used to detect the cell viability. Cell proliferation assay was conducted at distinct time intervals: 3, 5, and 7 days. RESULTS: The flow cytometry analysis verified the positive expression of mesenchymal cell surface antigen molecules (CD73, CD90, and CD105) and the absence of hematological markers (CD14, CD34, and CD45) in the DPSCs. The cells that treated with concentrations more than 0.5 mg/mL of Ca(OH2) and triple antibiotic paste (TAP) gave significant decrease in viability in comparison to the untreated cells (p < 0.05). Also, the cells treated with concentrations 50 and 25 µM of GA showed no significant difference compared with the untreated cells (p > 0.05), while concentrations 12.5 and 6.25 µM expressed a significant increase in viability compared with the untreated cells (p < 0.05). At 7 days, cells treated with the three different concentrations of GA (12.5, 25, and 50 µM) demonstrated a significant increase in cell density compared with Ca(OH)2 and TAP-treated cells (p < 0.05). CONCLUSION: Based upon the potential of GA on DPSCs proliferation compared with Ca(OH)2 and TAP, It is conceivable to acknowledge that GA could be used as an intracanal medicaments for revascularization process of necrotic immature teeth. CLINICAL SIGNIFICANCE: This study emphasizes the significance of assessing alternative root canal medicaments and their impact on the proliferation and viability of DPSCs. The results regarding GA, specifically its impact on the viability and growth of DPSCs, provide essential understanding for its potential application as an intracanal medicine. This study adds to the continuous endeavors in identifying safer and more efficient intracanal therapies, which are essential for improving patient outcomes in endodontic operations. How to cite this article: Alrashidi MA, Badawi MF, Elbeltagy MG, et al. The Effect of Glycyrrhizin on the Viability and Proliferation of Dental Pulp Stem Cells Compared to Intracanal Medicaments. J Contemp Dent Pract 2024;25(3):267-275.

Protective effect of dental pulp stem cells' conditioned medium against cisplatin-induced testicular damage in rats.

Cisplatin is a highly effective chemotherapy drug used to treat most solid tumors. However, one of its side effects is testicular toxicity, which can lead to fertility abnormalities. This study investigated the effectiveness of dental pulp mesenchymal stem cells conditioned medium (DPSC-CM) on cisplatin-induced testicular toxicity. In this study, 36 eight-week-old male Wistar rats were randomly divided into three groups equally (n = 12). Group 1 control "CTR", which received normal saline (0.5 ml) intraperitoneally (i.p), group 2 "Cis" which received an intraperitoneal dose of cisplatin (7 mg/kg), and group 3 "Cis+CM" which received an i.p injection of DPSC-CM (0.5 mg/kg) after cisplatin injection. Biochemical, histomorphometric, and histopathological studies were performed on the testis. Our results exhibited that cis administration led to a decline in total body weight, testis weight, diameter, and volume. A decrease in testosterone and IL-6 serum levels, as well as a decrease in IL-6 and TNFα levels, the activity of catalase and SOD enzymes, and an increase in MDA in testicular tissue were detected. Testicular tissue damage was associated with a significant decrease in tube diameter, germinal epithelium height, number of spermatogonia and Sertoli cells, along with a noticeable increase in basement membrane thickness, and perivascular fibrosis. DMSC-CM improved all the mentioned parameters. Taken together, our results demonstrated that DMSC-CM due to its antioxidant and anti-inflammatory properties, could be effective in reversing cisplatin-induced testicular toxicity.

Influence of dental bleaching on the pulp tissue: A systematic review of in vivo studies.

BACKGROUND: Although several studies indicate the harmful effects of bleaching on pulp tissue, the demand for this procedure using high concentrations of hydrogen peroxide (HP) is high. OBJECTIVES: To investigate the influence of bleaching on the pulp tissue. METHODS: Electronic searches were conducted (PubMed/MEDLINE, Scopus, Cochrane Library and grey literature) until February 2021. Only in vivo studies that evaluated the effects of HP and/or carbamide peroxide (CP) bleaching gels on the inflammatory response in the pulp tissue compared with a non-bleached group were included. Risk of bias was performed according to a modified Methodological Index for Non-Randomized Studies scale for human studies and the Systematic Review Centre for Laboratory Animal Experimentation's RoB tool for animal studies. Meta-analysis was unfeasible. RESULTS: Of the 1311 studies, 30 were eligible. Of these, 18 studies evaluated the inflammatory response in animal models. All these studies reported a moderate-to-strong inflammatory response in the superficial regions of pulp, characterized by cell disorganization and necrotic areas, particularly during the initial periods following exposure to 35%-38% HP, for 30-40 min. In the evaluation of human teeth across 11 studies, seven investigated inflammatory responses, with five observing significant inflammation in the pulp of bleached teeth. In terms of tertiary dentine deposition, 11 out of 12 studies noted its occurrence after bleaching with 35%-38% HP in long-term assessments. Additionally, three studies reported significant levels of osteocalcin/osteopontin at 2 or 10 days post-treatment. Other studies indicated an increase in pro-inflammatory cytokines ranging from immediately up to 10 days after bleaching. Studies using humans' teeth had a low risk of bias, whereas animal studies had a high risk of bias. DISCUSSION: Despite the heterogeneity in bleaching protocols among studies, High-concentrations of HP shows the potential to induce significant pulp damage. CONCLUSIONS: High-concentrations of bleaching gel increases inflammatory response and necrosis in the pulp tissue at short periods after bleaching, mainly in rat molars and in human incisors, in addition to greater hard tissue deposition over time. However, further well-described histological studies with long-term follow-up are encouraged due to the methodological limitations of these studies. REGISTRATION: PROSPERO (CRD42021230937).

Anti-inflammatory cerium-containing nano-scaled mesoporous bioactive glass for promoting regenerative capability of dental pulp cells.

AIMS: This study aimed to investigate the anti-inflammatory and odontoblastic effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on dental pulp cells as novel pulp-capping agents. METHODOLOGY: Ce-MBGNs were synthesized using a post-impregnation strategy based on the antioxidant properties of Ce ions and proposed the first use of Ce-MBGNs for pulp-capping application. The biocompatibility of Ce-MBGNs was analysed using the CCK-8 assay and apoptosis detection. Additionally, the reactive oxygen species (ROS) scavenging ability of Ce-MBGNs was measured using the 2,7-Dichlorofuorescin Diacetate (DCFH-DA) probe. The anti-inflammatory effect of Ce-MBGNs on THP-1 cells was further investigated using flow cytometry and quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the effect of Ce-MBGNs on the odontoblastic differentiation of the dental pulp cells (DPCs) was assessed by combined scratch assays, RT-qPCR, western blotting, immunocytochemistry, Alizarin Red S staining and tissue-nonspecific alkaline phosphatase staining. Analytically, the secretions of tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Ce-MBGNs were confirmed to effectively scavenge ROS in THP-1-derived macrophages and DPCs. Flow cytometry and RT-qPCR assays revealed that Ce-MBGNs significantly inhibited the M1 polarization of macrophages (Mφ). Furthermore, the protein levels of TNF-α and IL-1ß were downregulated in THP-1-derived macrophages after stimulation with Ce-MBGNs. With a step-forward virtue of promoting the odontoblastic differentiation of DPCs, we further confirmed that Ce-MBGNs could regulate the formation of a conductive immune microenvironment with respect to tissue repair in DPCs, which was mediated by macrophages. CONCLUSIONS: Ce-MBGNs protected cells from self-produced oxidative damage and exhibited excellent immunomodulatory and odontoblastic differentiation effects on DPCs. As a pulp-capping agent, this novel biomaterial can exert anti-inflammatory effects and promote restorative dentine regeneration in clinical treatment. We believe that this study will stimulate further correlative research on the development of advanced pulp-capping agents.

Ginsenoside Rb1 alleviates lipopolysaccharide-induced inflammation in human dental pulp cells via the PI3K/Akt, NF-κB, and MAPK signalling pathways.

AIM: Among numerous constituents of Panax ginseng, a constituent named Ginsenoside Rb1 (G-Rb1) has been studied to diminish inflammation associated with diseases. This study investigated the anti-inflammatory properties of G-Rb1 on human dental pulp cells (hDPCs) exposed to lipopolysaccharide (LPS) and aimed to determine the underlying molecular mechanisms. METHODOLOGY: The KEGG pathway analysis was performed after RNA sequencing in G-Rb1- and LPS-treated hDPCs. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used for the assessment of cell adhesion molecules and inflammatory cytokines. Statistical analysis was performed with one-way ANOVA and the Student-Newman-Keuls test. RESULTS: G-Rb1 did not exhibit any cytotoxicity within the range of concentrations tested. However, it affected the levels of TNF-α, IL-6 and IL-8, as these showed reduced levels with exposure to LPS. Additionally, less mRNA and protein expressions of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were shown. With the presence of G-Rb1, decreased levels of PI3K/Akt, phosphorylated IκBα and p65 were also observed. Furthermore, phosphorylated ERK and JNK by LPS were diminished within 15, 30 and 60 min of G-Rb1 exposure; however, the expression of non-phosphorylated ERK and JNK remained unchanged. CONCLUSIONS: G-Rb1 suppressed the LPS-induced increase of cell adhesion molecules and inflammatory cytokines, while also inhibiting PI3K/Akt, phosphorylation of NF-κB transcription factors, ERK and JNK of MAPK signalling in hDPCs.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells.

OBJECTIVE: To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1ß, in cultured human dental pulp cells. METHODOLOGY: Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1ß in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. RESULTS: Stimulation of the pulp cells with IL-1ß resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1ß (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1ß was also blocked by incubation with the extract. CONCLUSIONS: Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1ß in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Nano eggshell-based slurry as a direct pulp-capping material: In vitro characterization and histopathological assessment in an experimental animal model.

AIM: Pulp vitality is essential for tooth integrity. Following pulp exposure, choosing a suitable pulp-capping material is crucial to maintain pulp vitality. However, the reparative dentine bridge created by calcium hydroxide (Ca(OH)2 ) is generally porous and incomplete. The aim of the current study is to assess the in vitro and in vivo bioactivities of nano eggshell-based slurry (NES), using NES as a direct pulp-capping material, compared with Ca(OH)2 in rabbit animal model. METHODOLOGY: Nano eggshell powder (NE) was characterized for particle morphology, chemical composition and ion release. In vitro bioactivity was tested by immersion in simulated body fluid (SBF) for 7 days. For histopathological evaluation, 36 adult New Zealand rabbits (72 pulp exposures) were divided into nine groups (n = 8) according to the pulp-capping material (NES, Ca(OH)2 and no capping as negative control group) and the animals were sacrificed after 7, 14 or 28 days. The pulps of the two lower central incisors were exposed and then directly capped by Ca(OH)2 or NES or left untreated. The cavities were then sealed with glass ionomer cement. Teeth were collected for histopathological evaluation using an optical microscope. Pulp haemorrhage, inflammation, fibrosis and calcific bridge formation were assessed. Results were statistically analysed using anova and Tukey's tests. RESULTS: Nano eggshell particles were spherical with a 20 nm diameter and were composed mainly of calcite. Statistical analysis showed that there was a significant increase in the release of all investigated ions between days 1 and 28, except for copper. NES group showed a significantly higher release of all elements as compared to Ca(OH)2 . Environmental scanning electron microscope micrographs of NES incubated for 7 days in SBF showed the formation of HAp with a Ca/P ratio (1.686). For histopathological evaluation, the difference between groups was statistically significant. At day 28, 75% of the pulps of the Ca(OH)2 group showed mild calcific bridge in comparison with 100% moderate calcific bridge in the NES group. The NES group showed significantly less inflammation at days 7 and 28, and higher fibrosis at day 7 compared with Ca(OH)2 . CONCLUSIONS: Nano eggshell-based slurry represents a promising novel direct pulp-capping material with favourable pulp tissue response.

The Potential Application of Human Gingival Fibroblast-Conditioned Media in Pulp Regeneration: An In Vitro Study.

Regenerative endodontic treatment based on tissue engineering has recently gained interest in contemporary restorative dentistry. However, low survival rates and poor potential differentiation of stem cells could undermine the success rate of pulp regenerative therapy. Human gingival fibroblast-conditioned medium (hGF-CM) has been considered a potential therapy for tissue regeneration due to its stability in maintaining multiple factors essential for tissue regeneration compared to live cell transplantation. This study aimed to investigate the potency of hGF-CM on stem cells from human dental pulp (DPSC) in pulp regeneration. A series of experiments confirmed that hGF-CM contributes to a significant increase in proliferation, migration capability, and cell viability of DPSC after H2O2 exposure. Moreover, it has been proved to facilitate the odontogenic differentiation of DPSC via qRT-PCR, ALP (alkaline phosphatase), and ARS (Alizarin Red S) staining. It has been discovered that such highly upregulated odontogenesis is related to certain types of ECM proteins (collagen and laminin) from hGF-CM via proteomics. In addition, it is found that the ERK pathway is a key mechanism via inhibition assay based on RNA-seq result. These findings demonstrate that hGF-CM could be beneficial biomolecules for pulp regeneration.

Influence of violet LED associated or not with peroxide gel on inflammation, mineralization, and collagen fiber maturation in dentin and pulp tissue.

OBJECTIVES: To evaluate the influence of violet LED, associated or not with a 17.5% hydrogen peroxide (HP) bleaching gel, on inflammation, mineralization in pulp tissue, and collagen fiber maturation in dentin and pulp tissue. MATERIALS AND METHODS: The maxillary molars of eighty Wistar rats were distributed into four groups (n = 10): CONT - without treatment; HP - 30 min application of 17.5% HP; LED - 20 min application of violet LED; and HP+LED - application of PH and violet LED. Rats were euthanized and jaws were processed for histologic and immunohistochemical evaluation (IL-17, IL-23, and osteocalcin) and picrosirius red immediately after (T0), and at 7 (T1), 15 (T2), and 30 days (T3) post-treatment, with Wilcoxon, Mann-Whitney, paired T-test, and T-test (α = 0.05). RESULTS: HP and HP+LED presented necrosis and severe inflammatory infiltrate. When compared to CONT group, LED presented severe osteocalcin (OCN) immunostaining in T2 and less immature fibers in T2 and T3. CONCLUSION: The violet LED caused no severe damage to the pulp tissue, increased IL-17 and IL-23 expression in T0 when associated with HP, and had no influence on pulp tissue mineralization, besides accelerating the maturation of collagen fibers of dentin. CLINICAL RELEVANCE: Violet LED therapy induced no inflammation in the pulp tissue of rats and played no role in pulp tissue fibrosis, besides accelerating the maturation of dentin collagen fibers.

In vitro evaluation of injectable Tideglusib-loaded hyaluronic acid hydrogels incorporated with Rg1-loaded chitosan microspheres for vital pulp regeneration.

Injectable systems receive attention in endodontics due to the complicated and irregular anatomical structure of root canals. Here, injectable Tideglusib (Td)-loaded hyaluronic acid hydrogels (HAH) incorporated with Rg1-loaded chitosan microspheres (CSM) were developed for vital pulp regeneration, providing release of Td and Rg1 to trigger odontoblastic differentiation of human dental pulp stem cells (DPSC) by Td and vascularization of pulp by Rg1. The optimal concentrations were determined as 90 nM and 50 µg/mL for Td and Rg1, and loaded in HA and CSM in HAH, respectively. Odontogenic (COL1A1, ALP, OCN, Axin-2, DSPP, and DMP1) and angiogenic (VEGFA, VEGFR2, and eNOS) differentiation of DPSC cultured in the presence of hydrogels was shown at gene expression level. Our results suggest that our injectable hydrogel formulation has potential to improve strategies for vital pulp regeneration. In vivo evaluations are needed to test the feasibility and potential of these hydrogels for vital pulp regeneration.

Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration.

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

Simvastatin treatment promotes proliferation of human dental pulp stem cells via modulating PI3K/AKT/miR-9/KLF5 signalling pathway.

Simvastatin serves as an effective therapeutic potential in the treatment of dental disease via alternating proliferation of dental pulp stem cells. First, western-blot and real-time quantitative PCR were used to detect the effect of simvastatin or LY294002 on the expression levels of AKT, miR-9 and KLF5, or determine the effect of miR-9. Simvastatin, KLF5 and AKT significantly enhanced the proliferation of pulp stem cells, whilst this effect induced by simvastatin was suppressed by LY294002, AKT siRNA, KLF5 siRNA and miR-9, and simvastatin dose-dependently upregulated the expression of PI3K. Furthermore, simvastatin upregulated PI3K and p-AKT expression in a concentration-dependent manner. LY294002 abrogated the upregulation of p-AKT expression levels induced by simvastatin, and LY294002 induced the miR-9 expression and simvastatin dose-dependently inhibited the expression of miR-9, by contrast, LY294002 reduced the KLF5 expression and simvastatin dose-dependently promoted the expression of KLF5. And using computational analysis, KLF5 was found to be a candidate target gene of miR-9, and which was further verified using luciferase assay. Finally, the level of KLF5 in cells was much lower following the transfection with miR-9 and KLF5 siRNA, and the level of AKT mRNA in cells was significantly inhibited after transfection with AKT siRNA than control. These findings suggested simvastatin could promote the proliferation of pulp stem cells, possibly by suppressing the expression of miR-9 via activating the PI3K/AKT signalling pathway, and the downregulation of miR-9 upregulated the expression of its target gene, KLF5, which is directly responsible for the enhanced proliferation of pulp stem cells.

The protective effects of S14G-humanin (HNG) against lipopolysaccharide (LPS)- induced inflammatory response in human dental pulp cells (hDPCs) mediated by the TLR4/MyD88/NF-κB pathway.

Pulpitis is reported in large populations of patients and significantly impacts their normal life quality. It is reported that the lipopolysaccharide (LPS) in Gram-negative bacteria induces severe inflammation in dental pulp tissues. S14G-humanin is a derivative of humanin and has been recently confirmed to possess promising anti-inflammatory properties. The current study aims to explore the possibility of treating pulpitis with S14G-humanin. LPS-stimulated dental pulp cells (DPCs) were utilized to simulate an inflammatory state in the progression of pulpitis. We found the elevated expressions and production of interleukin- 6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage chemoattractant protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), upregulated Pentraxin 3 (PTX3) and activated oxidative stress in LPS-treated DPCs were all reversed by treatment with 50 and 100 µM S14G-humanin. In addition, the LPS-induced elevated expression levels of toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (Myd88), and activation of the IκBα/NF-κB signaling pathway in hDPCs were significantly repressed by treatment with S14G-humanin. Conclusively, we found that S14G-humanin protected LPS-treated hDPCs by inhibiting the TLR4/MyD88/NF-κB signaling pathway.

Interleukin-10 Modulates the Metabolism and Osteogenesis of Human Dental Pulp Stem Cells.

The osteogenic differentiation of mesenchymal stem cells (MSCs) is strongly related with the inflammatory microenvironment. The ability of osteogenic differentiation of MSCs is vital for the bone tissue engineering. Interleukin (IL)-10, a well-known anti-inflammatory factor, plays a key role in tissue repair. Dental pulp stem cells (DPSCs), with the advantage of convenience of extraction, are suitable for the bone tissue engineering. Therefore, it is meaning to explore the effects of IL-10 on the osteogenic differentiation of DPSCs. The proliferation activity of DPSCs were evaluated by MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay [Promega]) and real-time polymerase chain reaction (RT-PCR). The osteogenic differentiation of DPSCs were determined by Alizarin Red staining, RT-PCR, and alkaline phosphatase activity test. The glucose metabolism was detected by Mito Stress test and glycolysis assay. IL-10 (10 or 20 nM) could enhance the osteogenic differentiation of DPSCs and promoted the metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS), whereas IL-10 (5 and 50 nM) has no obvious effects on the osteogenic differentiation of DPSCs. The OXPHOS inhibitor restrained the promotion of osteogenic differentiation induced by IL-10. These findings show that IL-10 can promote the osteogenesis of DPSCs through the activation of OXPHOS, which provides a potential way for enhancing the osteogenic differentiation of DPSCs in bone tissue engineering.

H2O2 gel bleaching induces cytotoxicity and pain conduction in dental pulp stem cells via intracellular reactive oxygen species on enamel/dentin disc.

BACKGROUND: Bleaching is widely accepted for improving the appearance of discolored teeth; however, patient compliance is affected by bleaching-related complications, especially bleaching sensitivity. This study aimed to investigate the role of reactive oxygen species (ROS) in cytotoxicity and pain conduction activated by experimental tooth bleaching. METHODS: Dental pulp stem cells with or without N-acetyl-L-cysteine (NAC), an ROS scavenger, were cultured on the dentin side of the enamel/dentin disc. Subsequently, 15% (90 min) and 40% (30 min) bleaching gels were painted on the enamel surface. Cell viability, intracellular ROS, Ca2+, adenosine triphosphate (ATP), and extracellular ATP levels were evaluated using the Cell Counting Kit-8 assay, 2',7'-dichlorodihydrofluorescein diacetate, CellROX, fura-3AM fluorescence assay, and ATP measurement kit. The rat incisor model was used to evaluate in vivo effects after 0, 1, 3, 7, and 30 days of bleaching. Changes in gene and protein expression of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNFα), transient receptor potential ankyrin 1 (TRPA1), and Pannexin1 (PANX1) in dental pulp stem cells and pulp tissue were detected through RT-PCR, western blotting, and immunofluorescence. RESULTS: The bleaching gel suppressed dental pulp stem cell viability and extracellular ATP levels and increased intracellular ROS, Ca2+, and intracellular ATP levels. The mRNA and protein expression of IL-6, TNFα, TRPA1, and PANX1 were up-regulated in vitro and in vivo. Furthermore, the 40% gel had a stronger effect than the 15% gel, and NAC ameliorated the gel effects. CONCLUSIONS: Our findings suggest that bleaching gels induce cytotoxicity and pain conduction in dental pulp stem cells via intracellular ROS, which may provide a potential therapeutic target for alleviating tooth bleaching nociception.

TRPA1 triggers hyperalgesia and inflammation after tooth bleaching.

Hyperalgesia has become a major problem restricting the clinical application of tooth bleaching. We hypothesized that transient receptor potential ankyrin 1 (TRPA1), a pain conduction tunnel, plays a role in tooth hyperalgesia and inflammation after bleaching. Dental pulp stem cells were seeded on the dentin side of the disc, which was cut from the premolar buccal tissue, with 15% (90 min) or 40% (3 × 15 min) bleaching gel applied on the enamel side, and treated with or without a TRPA1 inhibitor. The bleaching gel stimulated intracellular reactive oxygen species, Ca2+, ATP, and extracellular ATP in a dose-dependent manner, and increased the mRNA and protein levels of hyperalgesia (TRPA1 and PANX1) and inflammation (TNFα and IL6) factors. This increment was adversely affected by TRPA1 inhibitor. In animal study, the protein levels of TRPA1 (P = 0.0006), PANX1 (P < 0.0001), and proliferation factors [PCNA (P < 0.0001) and Caspase 3 (P = 0.0066)] increased significantly after treated rat incisors with 15% and 40% bleaching gels as detected by immunohistochemistry. These results show that TRPA1 plays a critical role in sensitivity and inflammation after tooth bleaching, providing a solid foundation for further research on reducing the complications of tooth bleaching.

Dual antioxidant-photosensitizing hydrogel system: Cross-linking of chitosan with tannic acid for enhanced photodynamic efficacy.

Herein, a new antioxidant-photosensitizing hydrogel based on chitosan has been developed to control photodynamic therapy (PDT) activity in cancer treatment. In PDT, photosensitizers generate reactive oxygen species (ROS) during photochemical reactions, leading oxidative damage to cancer cells. However, high ROS levels are lethal to non-target healthy cells and tissues such as endothelial cells and blood cells. To mediate these drawbacks, we improved PDT with a natural polyphenolic antioxidant, Tannic acid (TA), to control the ROS level and minimize side effects through singlet oxygen (1O2) scavenging. In this work, chitosan-based hydrogels were designed using tannic acid as an antioxidant cross-linker and loaded with water-soluble N, N'-di-(l-alanine)-3,4,9,10-perylene tetracarboxylic diimide (PDI-Ala) as a photosensitizer. Our results showed that the hydrogel formed a three-dimensional (3D) microstructure with good mechanical strength and significant singlet oxygen production and antioxidant activity. In addition, the behavior of human melanoma cell line A375 and dental pulp stem cells (as normal cells) was compared and studied during an in vitro photodynamic treatment. Normal cells had a higher viability than cancer cells, indicating that the PDT is more effective on cancer cells than on normal cells. The new hydrogels could be applied as an effective new drug to control PDT performance.

Effect of treatment with resiniferatoxin in an experimental model of pulpal inflammatory in mice.

AIM: To evaluate whether treatment with resiniferatoxin (RTX) is capable of lowering the plasma levels of PGE2 and TNF-α, as well as histopathological parameters in inflammation of pulp tissue in a mouse experimental model. METHODOLOGY: Ten groups of six BALB/c mice were formed as follows: healthy group (HC ), healthy group treated with RTX (HRTX ), two groups with pulp inflammation at 14 and 18 hours (PI14 /PI18 ), six groups with pulpal inflammation plus treatment with Ibuprofen (IBU14 /IBU18 ), dexamethasone (DEX14 /DEX18 ) and resiniferatoxin (RTX14 /RTX18 ) at 14 and 18 hours, respectively. Pulpal inflammation was induced through occlusal exposure of the pulp of the maxillary first molar. The plasma levels of PGE2 and TNF-α and the histological parameters of the pulp tissue of the HC and HRTX groups were evaluated at the time of acquiring the animals. In the other groups, the plasma levels of PGE2 and TNF-α and the histopathological parameters were evaluated at 14 and 18 hours after pulp damage. Plasma levels of PGE2 and TNF-α were quantified by ELISA, and the histopathological parameters were evaluated by H/E staining. Statistical significance was determined by one-way analysis of variance (ANOVA) to test for overall differences between group means. RESULTS: A significant increase (*p < .05) in plasma levels of PGE2 and TNF-α occurred 14 and 18 hours after pulp damage. In addition, treatment with RTX significantly decreased (*p < .05) the plasma levels of PGE2 and TNF-α at 14 and 18 hours after pulp damage, as well as the infiltrate of inflammatory cells at 18 hours after pulp damage, similarly to treatment with ibuprofen and dexamethasone. CONCLUSION: It was possible to detect systemic levels of PGE2 and TNF-α at 14 and 18 hours after pulp damage. Likewise, treatment with RTX was associated with an anti-inflammatory effect similar to treatment with ibuprofen and dexamethasone. These findings place resiniferatoxin as a therapeutic alternative in the treatment of inflammatory diseases in Dentistry.

An In Vitro Study of the Effects of Crocin on the Modulation of DSPP, VEGF-A, HLA-G5, STAT3 and CD200 Expression in Human Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) have been recommended as promising candidate for cell-based therapeutic applications due to high potentials in tissue repair/regeneration and modulation of immune responses. The gene expression change strategy by natural plant enhancers is an available opportunity to improve the stemness properties of these cells. The objective of this research was the evaluation of Crocin effects (saffron plant's bioactive compound) on immunoregulation and tissue regeneration-related biomarkers expression in human DPSCs. Based on the results of cell viability assay, application of 400 µM and lower concentrations of Crocin had no toxic effects on DPSCs; however, the time-dependent cytotoxic effects were observed at higher concentrations. This study, probably for the first time, detected the surface expression of CD200 in DPSCs with a slight time-dependent upward trend and reported that treatment with Crocin could increase expression of this macromolecule up to many times over. Also, it revealed that this carotenoid significantly led to the time-dependent upregulation of dentin sialophosphoprotein, vascular endothelial growth factor A, human leukocyte antigen-G5, and signal transducer and activator of transcription-3 messenger ribonucleic acids (mRNAs); however, this significant upregulation for STAT3 occurred, followed by a remarkable reduction. The results of this study indicated that cell treatment with Crocin may be effective in improving the stemness capacities of DPSCs. Therefore, the study provided basis for more insights into the biological effects of Crocin on DPSCs that it may aid in the future improvement of mesenchymal stem cell-based therapies.