Hualing Sun

SIRT6 Regulates Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Partially via Suppressing the Nuclear Factor‐κB Signaling Pathway

Sirtuin 6 (SIRT6) is a NAD‐dependent deacetylase involved in lifespan regulation. To evaluate the effect of SIRT6 on osteogenesis, rat bone marrow mesenchymal stem cells (rBMSCs) with enhanced or reduced SIRT6 function were developed. We observed that SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt‐related transcription factor 2 (RUNX2), and osteocalcin, while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor‐κB (NF‐κB) transcriptional activity and upregulated the expression of acetyl‐NF‐κB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF‐κB inhibitor BAY 11–7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of collagen/chitosan/hydroxyapatite scaffold could significantly boost new bone formation in rat cranial critical‐sized defects, as determined by microcomputed tomography and histological examination. These data confirm that SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF‐κB signaling. Stem Cells 2014;32:1943–1955

Effect of desensitising paste containing 8% arginine and calcium carbonate on biofilm formation of Streptococcus mutans in vitro.

OBJECTIVES To evaluate the influence of desensitising paste containing 8% arginine and calcium carbonate (Ar-Ca) on biofilm formation on dentine. METHODS Dentine discs were cut from extracted third molars and divided into the following three groups: no treatment, pumice treatment and Ar-Ca treatment. Surface topography and roughness were examined using scanning electron microscopy (SEM) and non-contact 3D surface profiler. After sterilisation, samples were incubated with Streptococcus mutans (S. mutans) for 4 h, 24 h and 72 h. Bacterial adhesion and biofilm formation were analysed using SEM, whereas MTT and lactic acid production assays were used to analyse the metabolic activity of S. mutans. RESULTS After polishing with either pumice or Ar-Ca, the surfaces of the samples became smoother than in the control group. The Ra values of the three experimental groups decreased significantly to 0.43 μm, 0.3 μm and 0.26 μm, respectively. Compared to the control group, fewer bacteria adhered to the dentine surface in the Ar-Ca group, while biofilm thickness decreased significantly for both groups after incubating for 24 h and 72 h. MTT and lactic acid production levers also showed a significant reduction in the Ar-Ca group. CONCLUSIONS Ar-Ca appears to present antibiofilm efficacy and may provide a promising approach to combat bacterial infection in hypersensitive dentinal lesions. CLINICAL SIGNIFICANCE As a clinical application of desensitising polishing paste, the paste containing 8% arginine and calcium carbonate could also inhibit the biofilm formation effectively.

DDIT3 overexpression increases odontoblastic potential of human dental pulp cells

Human dental pulp cells (HDPCs) with multi‐potential differentiational capacity can undergo odontoblastic differentiation when stimulated with proinflammatory cytokines. However, factors linking proinflammatory stimuli and their odontoblastic differentiation have, as yet, not been completely understood. As an apoptotic transcription factor, DDIT3 plays a crucial role in the inflammatory reaction and in osteogenic differentiation. Thus, we hypothesized that DDIT3 may participate in odontoblastic differentiation of HDPCs.

Effect of silane pretreatment on the immediate bonding of universal adhesives to computer-aided design/computer-aided manufacturing lithium disilicate glass ceramics

The aim of this study was to investigate the effect of silane pretreatment on the universal adhesive bonding between lithium disilicate glass ceramic and composite resin. IPS e.max ceramic blocks etched with hydrofluoric acid were randomly assigned to one of eight groups treated with one of four universal adhesives (two silane-free adhesives and two silane-containing adhesives), each with or without silane pretreatment. Bonded specimens were stored in water for 24 h. The shear bond strength (SBS) of the ceramic-resin interface was measured to evaluate bond strength, and the debonded interface after the SBS test was analysed using field-emission scanning electron microscopy to determine failure mode. Light microscopy was performed to analyse microleakage and marginal sealing ability. Silane pretreatment significantly and positively influenced SBS and marginal sealing ability. For all the universal adhesive groups, SBS increased and the percentage of microleakage decreased after the pretreatment. Without the pretreatment, SBS and the percentage of microleakage were not significantly different between the silane-containing universal adhesive groups and the silane-free groups. Cohesive failure was the main fracture pattern. The results suggest that additional silane pretreatment can effectively improve the bonding strength and marginal sealing of adhesives to lithium disilicate glass ceramics. The bonding performance of silane-containing universal adhesives without pretreatment is similar to that of silane-free adhesives.

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.