Shuheng Huang

NLRP3 Is Expressed in Human Dental Pulp Cells and Tissues

INTRODUCTION One of the best-characterized Nod-like receptor (NLR) family members is pyrin domain containing 3 (NLRP3). Intracellular NLRP3 is the most versatile innate immune receptor. On activation, NLRP3 assembles into a multiprotein complex, termed an inflammasome, which regulates the secretion and bioactivity of interleukin-1 family cytokines. NLRP3 has broad specificity for mediating an immune response to a wide range of microbial stimuli or danger signals. Therefore, we hypothesize that NLRP3 plays an essential role in the detection of bacterial pathogens and the initiation of inflammation within the dental pulp. Thus, the aim of this study was to evaluate the expression of NLRP3 in normal human dental pulp cells (HDPCs) and pulp tissues. METHODS Pulp tissues were collected from freshly extracted human third molars, and HDPCs were prepared from the explants of normal dental pulp tissues. Reverse transcription-polymerase chain reaction and Western blotting were performed to detect the levels of NLRP3 mRNA and protein, respectively. In addition, immunohistochemical staining was used to determine the distribution of NLRP3 in pulp tissues. RESULTS Normal human dental pulp tissues displayed high levels of NLRP3 mRNA and protein. NLRP3 proteins were principally expressed in odontoblasts and some pulp vascular endothelial cells. Moreover, HDPCs also expressed NLRP3 but at a relatively low level in comparison with that of dental pulp tissues. CONCLUSIONS The expression of NLRP3 in HDPCs and pulp tissues suggests that NLRP3-mediated signaling pathways may play an important role in dental immune defense.

lncRNA DANCR suppresses odontoblast-like differentiation of human dental pulp cells by inhibiting wnt/β-catenin pathway.

Long noncoding RNAs (lncRNAs) have recently emerged as an important class of regulatory molecules in diverse biological processes, although lncRNA involvement in the odontoblast-like differentiation of human dental pulp cells (hDPCs) is poorly understood. We investigate the expression of lncRNAs in this differentiation and explore their underlying role and the involved mechanism. Integrated comparative lncRNA microarray profiling was used to examine lncRNA expression during this differentiation. The differential expression of lncRNAs was validated by quantitative real-time reverse transcription plus the polymerase chain reaction. Differential lncRNA overexpression was performed with an adenoviral vector and the role and mechanism was then investigated in odontoblast-like differentiation. We identified 139 differentially expressed lncRNAs during this differentiation. Among them, five lncRNAs differentially expressed in microarray analysis were validated. Notably, lncRNA DANCR expression was significantly downregulated during hDPC differentiation to odontoblast-like cells in a time-dependent manner. Moreover, lncRNA DANCR overexpression blocked mineralized nodule formation and the expression of DSPP and DMP-1 in hDPCs after 14 days of odontogenic induction. Importantly, the upregulation of DANCR significantly decreased the expression levels of p-GSK-3β and β-catenin expression indicating that lncRNA DANCR can inhibit the activation of the Wnt/β-catenin signal pathway during the odontoblast-like differentiation of hDPCs. Thus, the modulation of Wnt/β-catenin signaling by lncRNA DANCR represents a potential therapeutic option for reparative dentin formation and regenerative endodontics.

Effect of Enterococcus faecalis Lipoteichoic Acid on Apoptosis in Human Osteoblast-like Cells

INTRODUCTION Enterococcus faecalis is commonly detected in persistent apical periodontitis characterized by unimproved periradicular bone resorption. The aim of the present study was to examine the effect of lipoteichoic acid (LTA), a major virulence factor of E. faecalis, on apoptosis of osteoblasts. METHODS Human osteoblast-like MG63 cells were treated with LTA from E. faecalis at a series of concentrations for 48 hours. The proliferation of the MG63 cells was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide assay. To examine the apoptosis, the LTA-treated cells were analyzed by flow cytometry by using annexin V-fluorescein isothiocyanate and propidium iodide double staining. Hoechst 33258 staining was performed to observe the morphologic changes of the cells. To investigate the apoptosis at the molecular level, the protein levels of Bcl-2 and Bax were determined by Western blot. Meanwhile, the caspase-3 activity was detected with caspase colorimetric protease assay. RESULTS The proliferation of MG63 cells was inhibited by E. faecalis LTA in a dose-dependent manner. Flow cytometry assay indicated that LTA had a stimulating effect on MG63 cell apoptosis. Typical morphologies of apoptotic cells were observed under fluorescence microscope. Furthermore, the cell apoptosis was confirmed by (1) the down-regulation of the antiapoptotic protein (Bcl-2), (2) the up-regulation of the proapoptotic protein (Bax), and (3) the elevated caspase-3 activity. CONCLUSIONS LTA of E. faecalis could inhibit the proliferation and induce apoptosis of human osteoblast-like MG63 cells.

JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells

Abstract Bone morphogenetic protein-2 (BMP-2) is a multi-functional growth factor belonging to the transforming growth factor β superfamily that has a broad range of activities that affect many different cell types. BMP-2 induces odontoblastic differentiation of human dental pulp cells (DPCs), but the underlying mechanism remains unclear. In this study, we investigated the potential role of the JNK mitogen-activated protein kinases (MAPK) pathway in BMP-2-induced odontoblastic differentiation of DPCs. The levels of phosphorylated and unphosphorylated JNK MAPK were quantified by Western blot analysis following treatment with BMP-2 and the JNK inhibitor SP600125. The role of JNK MAPK in the BMP-2-induced odontoblastic differentiation of DPCs was determined by measuring alkaline phosphatase (ALP) activity and by examining the expression of odontoblastic markers using quantitative real-time polymerase chain reaction analysis. The effect of JNK MAPK silencing on odontoblastic differentiation was also investigated. BMP-2 upregulated the phosphorylation of JNK in DPCs in a dose- and time-dependent manner. Early markers of odontoblastic differentiation, including ALP activity, osteopontin and dentin matrix protein-1, were not inhibited by the JNK inhibitor. However, the JNK inhibitor, SP600125, significantly inhibited late-stage differentiation of odontoblasts, including the gene expression of osteocalcin, dentin sialophosphoprotein and bone sialoprotein, and also reduced the formation of mineralized nodules in BMP-2-treated DPCs. Consistent with this observation, silencing of JNK MAPK also decreased late-stage odontoblastic differentiation. Taken together, these findings suggest that JNK activity is required for late-stage odontoblastic differentiation induced by BMP-2.

Absent in Melanoma 2 (AIM2) Expressed in Human Dental Pulp Mediates IL-1β Secretion in Response to Cytoplasmic DNA

The inflammasome has been determined to play an important role in inflammatory diseases in recent years. Absent in melanoma 2 (AIM2), an inflammasome that recognizes cytoplasmic DNA, has recently been identified as a critical regulator of immune responses. In this study, we explored whether AIM2 was expressed in human dental pulp and defined the role of AIM2 in regulating interleukin (IL)-1β secretion. We demonstrated that AIM2 was only detected in the odontoblast layer of healthy dental pulp, whereas strong expression was observed in inflamed dental pulp. Stimulation with interferon gamma (IFN-γ) and cytoplasmic DNA significantly activated the AIM2 inflammasome and increased IL-1β secretion in human dental pulp cells (HDPCs) in a time- and dose-dependent manner. Moreover, the knockdown of AIM2 downregulated both cleaved-caspase-1 expression and IL-1β release in HDPCs. These results suggest that AIM2 expressed in human dental pulp plays an important role in the immune defense by activating the inflammasome signaling pathway.

Genetic modification to induce CXCR2 overexpression in mesenchymal stem cells enhances treatment benefits in radiation-induced oral mucositis

Radiation-induced oral mucositis affects patient quality of life and reduces tolerance to cancer therapy. Unfortunately, traditional treatments are insufficient for the treatment of mucositis and might elicit severe side effects. Due to their immunomodulatory and anti-inflammatory properties, the transplantation of mesenchymal stem cells (MSCs) is a potential therapeutic strategy for mucositis. However, systemically infused MSCs rarely reach inflamed sites, impacting their clinical efficacy. Previous studies have demonstrated that chemokine axes play an important role in MSC targeting. By systematically evaluating the expression patterns of chemokines in radiation/chemical-induced oral mucositis, we found that CXCL2 was highly expressed, whereas cultured MSCs negligibly express the CXCL2 receptor CXCR2. Thus, we explored the potential therapeutic benefits of the transplantation of CXCR2-overexpressing MSCs (MSCsCXCR2) for mucositis treatment. Indeed, MSCsCXCR2 exhibited enhanced targeting ability to the inflamed mucosa in radiation/chemical-induced oral mucositis mouse models. Furthermore, we found that MSCCXCR2 transplantation accelerated ulcer healing by suppressing the production of pro-inflammatory chemokines and radiogenic reactive oxygen species (ROS). Altogether, these findings indicate that CXCR2 overexpression in MSCs accelerates ulcer healing, providing new insights into cell-based therapy for radiation/chemical-induced oral mucositis.

The Role of Transient Receptor Potential Cation Channel, Subfamily C, Member 1 in the Odontoblast-like Differentiation of Human Dental Pulp Cells.

Introduction: Calcium ions (Ca2+) actively participate in reparative dentin formation by promoting cellular proliferation and differentiation of human dental pulp cells (hDPCs). Transient receptor potential cation channel, subfamily C, member 1 (TRPC1) activates Ca2+ entry upon store depletion in a variety of cell types. However, the function of TRPC1 in hDPCs has not been reported. Therefore, we aimed to analyze the role of TRPC1 in hDPCs undergoing odontoblast‐like differentiation. Methods: Immunohistochemical staining was used to determine the distribution of TRPC1 in pulp tissues. Western blot analysis was used to detect the protein level of TRPC1 in the odontoblast‐like differentiation of hDPCs. Knockdown of TRPC1 was performed with an adenoviral vector to evaluate the role of TRPC1 in hDPCs during odontoblast‐like differentiation. Results: The results showed that TRPC1 was highly expressed in the cytoplasm of dental pulp cells, especially in the odontoblast layer of the healthy pulp. Moreover, the protein level of TRPC1 increased in a time‐dependent manner during the odontoblast‐like differentiation of hDPCs. Importantly, knockdown of TRPC1 attenuated the process of odontoblast‐like differentiation as indicated by the reduction in mineralized nodules and the down‐regulation of dentin sialophosphoprotein and dentin matrix protein 1. Moreover, knockdown of TRPC1 decreased Ca2+ entry to the cytoplasm of hDPCs. Conclusions: Our data indicated a pivotal role of TRPC1 in the odontoblastlike differentiation of hDPCs, which may be a therapeutic target to enhance reparative dentin formation. Highlights:Transient receptor potential cation channel, subfamily C, member 1 (TRPC1) exerted important roles in controlling odontoblastlike differentiation.Down‐regulation of TRPC1 attenuated odontoblastlike differentiation.Knockdown of TRPC1 decreased calcium ion entry to the cytoplasm of human dental pulp cells.TRPC1 may be a potential therapeutic target for regenerative endodontics.

The antibacterial, cytotoxic, and flexural properties of a composite resin containing a quaternary ammonium monomer

Statement of problem. The use of composite resin to restore teeth has increased substantially during the last decades. However, secondary caries and the fracture of restorations are the leading reasons for clinical restoration failure. Mechanically strong composite resins with caries‐inhibition capabilities are needed. Although antibacterial dimethacrylate quaternary ammonium monomers have been synthesized, composite resin containing dimethacrylate quaternary ammonium monomers and glass fillers has rarely been reported. Purpose. The purpose of this in vitro study was to evaluate the possibility of the clinical use of an experimental composite resin containing urethane dimethacrylate quaternary ammonium compound (UDMQA‐12) by investigating its antibacterial activity, cytotoxicity, flexural strength, and flexural modulus. Material and methods. Antibacterial activity against Streptococcus mutans was investigated by means of direct contact test. The antibacterial activity of specimens after water immersion and saliva treatment was also tested. These were compared with a commercially available composite resin, Z250, and a glass ionomer cement, Fuji VII. Effects of the eluent from the experimental composite resin on the metabolic activity of human dental pulp cells were quantified. Disks of 1 mm in thickness and 15 mm in diameter were used in the antibacterial and cytotoxic tests. Flexural strength and flexural modulus were measured with a 3‐point bend test with bars of 2×2×25 mm. Three commercially available composite resins (Filtek Z250, G‐aenial Anterior, and G‐aenial Posterior) were used as controls in the flexural test. Results. Bacterial growth was inhibited on the experimental composite resin. After water immersion or saliva treatment, the experimental composite resin showed significant antibacterial effect compared with the conventional composite resin (P<.05). No significant difference was found in cytotoxicity between the experimental composite resin and the conventional composite resin (P > .05), and a significantly higher cytotoxicity was shown by glass ionomer cement compared with the experimental composite resin and the conventional composite resin (P<.05). The conventional composite resin had the highest flexural strength and flexural modulus (P<.05), followed by the experimental composite resin, then G‐ænial Posterior and G‐ænial Anterior. Conclusions. The antibacterial experimental composite resin was biocompatible and had mechanical properties similar to those of some commercially available composite resins. It might, therefore, be useful in preventing the occurrence of secondary caries.

AIM2 Inflammasome Is Critical for dsDNA-Induced IL-1β Secretion in Human Dental Pulp Cells

The AIM2 inflammasome pathway has been determined to play an important role in cellular immune defense against bacterial and viral infections; however, its function and regulatory mechanism in human dental pulp cells (HDPCs) during pulpitis remains poorly understood. In this study, we explored whether the AIM2 inflammasome pathway was activated in HDPCs in response to dsDNA and defined its role in regulating IL-1β secretion. We demonstrated that stimulation with IFN-γ and cytoplasmic DNA significantly activated the AIM2 inflammasome and increased IL-1β secretion in HDPCs. Moreover, AIM2 overexpression significantly up-regulated both cleaved Caspase-1 expression and IL-1β release in HDPCs, while suppression of ASC and Caspase-1 resulted in down-regulation of cleaved Caspase-1 and IL-1β secretion. These results suggest that Caspase-1-dependent IL-1β processing and secretion require the AIM2 inflammasome pathway in HDPCs and that the AIM2 inflammasome pathway is critical for regulation of the dental pulp immune response.

Role of transient receptor potential channel 6 in the odontogenic differentiation of human dental pulp cells

Pulp capping is a restorative technique employed in an attempt to maintain pulpal vitality and generate reparative dentin. Ca2+ released from capping materials is suggested to promote reparative dentin formation. Transient receptor potential channel 6 (TRPC6) is a receptor-operated Ca2+ channel that serves an important role in Ca2+ influx in the majority of non-excitable cells, and influences the calcium signaling and cell respond. Therefore, the purpose of the present study was to gain an insight into the role of TRPC6 in the odontoblastic differentiation of human dental pulp cells (HDPCs). Human dental pulp tissues and HDPCs were obtained from healthy third molars. By immunohistochemical staining, TRPC6 was observed to be highly expressed in the dental pulp tissue, particularly in the odontoblast layer. In addition, the protein level of TRPC6 was increased in a time-dependent manner during odontogenic differentiation of HDPCs. Downregulation of TRPC6 by a lentivirus vector containing TRPC6 shRNA inhibited the process of odontogenic differentiation in HDPCs. In conclusion, the current data demonstrated that TRPC6 served a significant role in the odontogenic differentiation of HDPCs, suggesting it may be a promising therapeutic target in regenerative endodontics.