Fangfang Song

Expression of Pannexin3 in human odontoblast-like cells and its hemichannel function in mediating ATP release

OBJECTIVE The aim of this study is to investigate the expression of pannexin3 (Panx3) in human odontoblast-like cells (hOBs) and its hemichannel function in mediating ATP release. METHODS RT-PCR and immunofluorescence analysis were used to detect the expression of pannexins (Panxs) in human dental pulp tissue and cultured cells. To determine the role of Panx3 in ATP release, hOBs were infected with Panx3-overexpression lentivirus, Panx3-shRNA lentivirus or control lentivirus and then stimulated with cold buffer. Intracellular ATP was monitored using quinacrine, and then semi-quantitatively analyzed. In the meantime, the ATP release was quantitatively analyzed using the bioluminescence method when the cells were exposed to cold stimulus. RESULTS Panx3 mRNA and protein were found in dental pulp tissue and cultured cells. Upon cold stimulus, intracellular ATP was released into the extracellular space. Overexpression of Panx3 accelerated ATP release, whereas inhibition of Panx3 suppressed this process. CONCLUSION Panx3 hemichannel is expressed in human odontoblast-like cells and mediates ATP release into the extracellular space.

Pannexin3 inhibits TNF-α-induced inflammatory response by suppressing NF-κB signalling pathway in human dental pulp cells

Human dental pulp cells (HDPCs) play a crucial role in dental pulp inflammation. Pannexin 3 (Panx3), a member of Panxs (Pannexins), has been recently found to be involved in inflammation. However, the mechanism of Panx3 in human dental pulp inflammation remains unclear. In this study, the role of Panx3 in inflammatory response was firstly explored, and its potential mechanism was proposed. Immunohistochemical staining showed that Panx3 levels were diminished in inflamed human and rat dental pulp tissues. In vitro, Panx3 expression was significantly down‐regulated in HDPCs following a TNF‐α challenge in a concentration‐dependent way, which reached the lowest level at 10 ng/ml of TNF‐α. Such decrease could be reversed by MG132, a proteasome inhibitor. Unlike MG132, BAY 11‐7082, a NF‐κB inhibitor, even reinforced the inhibitory effect of TNF‐α. Quantitative real‐time PCR (qRT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) were used to investigate the role of Panx3 in inflammatory response of HDPCs. TNF‐α‐induced pro‐inflammatory cytokines, interleukin (IL)‐1β and IL‐6, were significantly lessened when Panx3 was overexpressed in HDPCs. Conversely, Panx3 knockdown exacerbated the expression of pro‐inflammatory cytokines. Moreover, Western blot, dual‐luciferase reporter assay, immunofluorescence staining, qRT‐PCR and ELISA results showed that Panx3 participated in dental pulp inflammation in a NF‐κB‐dependent manner. These findings suggested that Panx3 has a defensive role in dental pulp inflammation, serving as a potential target to be exploited for the intervention of human dental pulp inflammation.

Yes-associated protein 1 promotes the differentiation and mineralization of cementoblast†

Yes‐associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt‐related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1‐knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad‐dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP‐stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad‐dependent BMP and Erk1/2 signaling pathways.

YAP1 negatively regulates chondrocyte differentiation partly by activating the β-catenin signaling pathway

YAP1 (Yes-associated protein 1) transcriptional coactivator is a downstream gene of the Hippo signaling pathway, which controls cell proliferation and differentiation. YAP1 plays a significant role in the regulation of cartilage and bone development. However, the molecular mechanism by which YAP1 regulates chondrocyte differentiation remains to be elucidated. Immunofluorescent staining was used to visualize the localization of YAP1 expression in the mouse chondroprogenitor ATDC5 cell line. ATDC5 cells with lentivirus-vector-mediated YAP1 overexpression and knockdown were established. The differentiation abilities were examined by real-time quantitative PCR and two staining methods The expression levels of sex-determining region Y-type high mobility group box protein (SOX9) and key proteins in the Wnt/β-catenin pathway were analyzed by Western blot. The Dickkopf-1 (Dkk1) and small interfering RNA (siRNA) of β-catenin were used for further study. The YAP1 protein was mainly expressed in the nucleus of ATDC5 cells. YAP1 overexpression enhanced chondrocyte proliferation but inhibited chondrocyte differentiation, which were contrary to the findings of the YAP1-knockdown group. Moreover, YAP1 overexpression activated Wnt/β-catenin signaling pathway. Treatment with exogenous DKK1 and β-catenin siRNA partially recaptured the effects of YAP1 overexpression on ATDC5 cell differentiation. Taken together, our study suggested that YAP1 attenuated ATDC5 cell chondrogenic and hypertrophic differentiation. We also demonstrated that YAP1 exerted its effect on the chondrocyte differentiation by activating the Wnt/β-catenin signaling pathway.

Ddit3 suppresses the differentiation of mouse chondroprogenitor cells

Endochondral ossification is an essential skeletal development process which is strongly linked to chondrocyte differentiation. DNA damage-inducible transcript 3 (Ddit3), a member of the CCAAT/enhancer-binding protein family of transcription factors, is highly expressed in the cartilage plate. However, the role of DNA damage-inducible transcript 3 in chondrocyte differentiation remains to be investigated. Immunofluorescent staining was used to detect Ddit3 expression in the mouse growth plate and in the mouse chondroprogenitor cell line ATDC5. A lentivirus system was employed to overexpress Ddit3 and silence its endogenous expression in ATDC5 cells. The differentiation abilities of ATDC5 cells were examined through quantitative reverse transcription polymerase chain reaction (qRT-PCR) and chondrogenic and hypertrophic-related staining. Western blot analysis was performed to detect the protein expression of sex-determining region Y-type high-mobility group box 9 and CCAAT/enhancer-binding protein β. Ddit3 was expressed in the proliferative and hypertrophic zones of the mouse growth plate. Ddit3 knockdown significantly enhanced the expression of chondrogenic and hypertrophic markers, whereas Ddit3 overexpression decreased the expression of these markers. This finding was also evidenced by Alcian blue staining, proteoglycan synthesis and alkaline phosphatase assay. Additionally, Ddit3 down-regulation significantly led to Sox9 up-regulation. These results suggest that Ddit3 suppresses the differentiation of ATDC5 cells. The function of Ddit3 might partially be regulated by Sox9 expression during chondrogenic and hypertrophic differentiation.

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.

Influence of naringenin on the biofilm formation of Streptococcus mutans

OBJECTIVES To evaluate the effect of naringenin on the biofilm formation of Streptococcus mutans (S. mutans), and to investigate its mechanisms of action and biological toxicity. METHODS Minimum inhibitory concentrations, growth curves, and biofilm inhibition rates of naringenin were determined to assess its antimicrobial effect on S. mutans. The morphology of S. mutans and the structure of biofilm were observed by FESEM and CLSM. Bacterial aggregation, bacterial surface hydrophobicity, and real-time PCR for gtfB, gtfC, comD, comE, and luxS mRNA expression were assessed to preliminarily investigate the mechanisms of action. MTT test using human dental pulp cells (HDPCs) was also performed to investigate cytotoxicity. RESULTS The S.mutans growth curves, FESEM, CLSM showed that both 100 and 200 μg/mL of naringenin obviously inhibited S. mutans growth and biofilm formation, increased S. mutans surface hydrophobicity, reduced bacterial aggregation, and downregulated the mRNA expression of gtfB, gtfC, comD, comE, and luxS. However, naringenin at 200 μg/mL slightly decreased the growths of HDPCs compared with 100 μg/mL. CONCLUSION Naringenin at 100 and 200 μg/mL suppressed the second (bacterial adhesion) and third stages (biofilm maturation) of S. mutans biofilm formation. CLINICAL SIGNIFICANCE Naringenin is promising for dental clinic promotion to prevent the biofilm formation of S. mutans, serving as a safe anti-caries agent at an appropriate concentration.

Establishment of 3D culture and induction of osteogenic differentiation of pre-osteoblasts using wet-collected aligned scaffolds

Aligned fibrous scaffolds have attracted much interest in bone tissue engineering, because they are supposed to induce osteogenic differentiation. For the first time, aligned silk fibroin nanofibres were loosely packed using a novel wet-collection electrospinning method. Moreover, three-dimensional (3D) culture of MC3T3-E1 pre-osteoblasts was established on these fibrous scaffolds. Physicochemical properties of the scaffolds and the behaviour of MC3T3-E1 pre-osteoblasts on the scaffolds were analysed and compared with scaffolds obtained using traditional method. Ethanol bath improved the uniformity and alignment of the fibres and increased the thickness and porosity of the scaffolds. Structures of the fibres were well maintained after immediate crosslinking in ethanol. Cells on the wet-collected scaffolds exhibited more ordered arrangement and elongated morphology as well as faster and deeper infiltration. The ordered infiltration resulted in the establishment of the 3D culture of cells, promoting proliferation and osteogenic differentiation of the pre-osteoblasts. Thus, the wet-collected aligned scaffolds with improved topographical and physicochemical properties presents significant potential application in bone regeneration.

Application of electrophoretic deposition to occlude dentinal tubules in vitro

OBJECTIVES This study aims to apply electrophoretic deposition (EPD) for occlusion of dentinal tubules in vitro and investigate its effect on tubule occlusion and shear bond strength (SBS). METHODS Charged mesoporous silica nanoparticles (MSNs) were synthesized and characterized through field-emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared (FT-IR) spectroscopy analyses. Thirty-nine sensitive dentin specimens were modeled and assigned randomly to three groups with different treatments (n = 13 each): group 1, immersion in the MSN suspension; and groups 2 and 3, anodic EPD with the specimen on the negative and positive electrode respectively. The effect of dentinal tubule occlusion was evaluated by dentin permeability test (n = 10 each) and FESEM examination (n = 3 each). Moreover, 18 specimens were grouped (n = 6 each) and treated in the same method. A resin stick was bonded onto each of the specimen using a self-etch adhesive (single bond universal) for SBS testing. RESULTS Negatively-charged MSNs were synthesized and characterized as small and well-dispersed particles. After the EPD treatment (group 3), the dentinal tubules were effectively occluded by MSNs, which infiltrated into the tubules at a depth of approximately 7-8 μm and tightly associated with the tubular inwalls. SBS was not significantly different among the three groups (P > 0.05). CONCLUSIONS Synthesized MSNs were deposited into dentinal tubules by EPD treatment without compromising dentin bond strength. CLINICAL SIGNIFICANCE Application of EPD is a new approach for occlusion of dentinal tubules and exhibits potential in the study of dentin hypersensitivity.

IRX5 promotes NF‐κB signalling to increase proliferation, migration and invasion via OPN in tongue squamous cell carcinoma

Iroquois homeobox gene 5 (Irx5) is a highly conserved member of the Iroquois homeobox gene family. Members of this family play distinct and overlapping roles in normal embryonic cell patterning and development of malignancies. In this study, we observed that IRX5 was abnormally abundant in tongue squamous cell carcinoma (TSCC) tissues and cell lines. We used gain‐ and loss‐of‐function methods to overexpress and knockdown IRX5 expression in the TSCC cell line CAL27. Our results elucidated that elevated levels of IRX5 promoted proliferation, migration and invasion of TSCC cells, whereas stable or transient knockdown of IRX5 expression suppressed TSCC cell proliferation, migration and invasion. As a transcription factor, IRX5 performed this function by targeting osteopontin (OPN) promoter and activating the NF‐κB pathway. Finally, studies in xenograft tumour model showed that IRX5 significantly enhanced OPN expression and promoted tumour growth. Taken together, our study elucidates a promotive effect of IRX5 in TSCC through the connection with OPN. These findings reveal the new molecular mechanism of TSCC, which may potentiate its use as a novel molecular therapy target for TSCC.