Deferoxamine-Induced Migration and Odontoblast Differentiation via ROS-Dependent Autophagy in Dental Pulp Stem Cells.

BACKGROUND/AIMS: As a vital degradation and recycling system, autophagy plays an essential role in regulating the differentiation of stem cells. We previously showed that iron chelator deferoxamine (DFO) could promote the repair ability of dental pulp stem cells (DPSCs). Here, we investigated the effect of DFO in autophagy and the role of autophagy in regulating the migration and odontoblast differentiation of DPSCs. METHODS: Transmission electron microscopy, immunofluorescence staining and western blotting were performed to evaluate the autophagic activity of DPSCs. Transmigration assay, alkaline phosphatase staining/activity, alizarin red S staining and quantitative PCR were performed to examine the migration and odontoblast differentiation of DPSCs. Reactive oxygen species (ROS) levels and the effects of ROS scavenger in autophagy induction were also detected. Autophagy inhibitors (3-MA and bafilomycin A1) and lentiviral vectors carrying ATG5 shRNA sequences were used for autophagy inhibition. RESULTS: Early exposure to DFO promoted the mineralization of DPSCs and increased autophagic activity. Autophagy inhibition suppressed DFO-induced DPSC migration and odontoblast differentiation. Furthermore, DFO treatment could induce autophagy partly through hypoxia-inducible factor 1α/B cell lymphoma 2/adenovirus E1B 19K-interacting protein 3 (HIF-1α/BNIP3) pathway in a ROS-dependent manner. CONCLUSION: DFO increased DPSC migration and differentiation, which might be modulated through ROS-induced autophagy.

Progress risk assessment of oral premalignant lesions with saliva miRNA analysis.

BACKGROUND: Oral cancer develops through multi-stages: from normal to mild (low grade) dysplasia (LGD), moderate dysplasia, and severe (high grade) dysplasia (HGD), to carcinoma in situ (CIS) and finally invasive oral squamous cell carcinomas (OSCC). Clinical and histological assessments are not reliable in predicting which precursor lesions will progress. The aim of this study was to assess the potential of a noninvasive approach to assess progress risk of oral precancerous lesions. METHODS: We first used microRNA microarray to profile progressing LGD oral premaligant lesions (OPLs) from non-progressing LGD OPLs in order to explore the possible microRNAs deregulated in low grade OPLs which later progressed to HGD or OSCC. We then used RT-qPCR to detect miRNA targets from the microarray results in saliva samples of these patients. RESULTS: We identified a specific miRNA signature that is aberrantly expressed in progressing oral LGD leukoplakias. Similar expression patterns were detected in saliva samples from these patients. CONCLUSIONS: These results show promise for using saliva miRNA signature for monitoring of cancer precursor lesions and early detection of disease progression.

Periodontitis exacerbates atherosclerosis through Fusobacterium nucleatum-promoted hepatic glycolysis and lipogenesis.

AIMS: Positive associations between periodontitis (PD) and atherosclerosis have been established, but the causality and mechanisms are not clear. We aimed to explore the causal roles of PD in atherosclerosis and dissect the underlying mechanisms. METHODS AND RESULTS: A mouse model of PD was established by ligation of molars in combination with application of subgingival plaques collected from PD patients and then combined with atherosclerosis model induced by treating atheroprone mice with a high-cholesterol diet (HCD). PD significantly aggravated atherosclerosis in HCD-fed atheroprone mice, including increased en face plaque areas in whole aortas and lesion size at aortic roots. PD also increased circulating levels of triglycerides and cholesterol, hepatic levels of cholesterol, and hepatic expression of rate-limiting enzymes for lipogenesis. Using 16S ribosomal RNA (rRNA) gene sequencing, Fusobacterium nucleatum was identified as the most enriched PD-associated pathobiont that is present in both the oral cavity and livers. Co-culture experiments demonstrated that F. nucleatum directly stimulated lipid biosynthesis in primary mouse hepatocytes. Moreover, oral inoculation of F. nucleatum markedly elevated plasma levels of triglycerides and cholesterol and promoted atherogenesis in HCD-fed ApoE-/- mice. Results of RNA-seq and Seahorse assay indicated that F. nucleatum activated glycolysis, inhibition of which by 2-deoxyglucose in turn suppressed F. nucleatum-induced lipogenesis in hepatocytes. Finally, interrogation of the molecular mechanisms revealed that F. nucleatum-induced glycolysis and lipogenesis by activating PI3K/Akt/mTOR signalling pathway in hepatocytes. CONCLUSIONS: PD exacerbates atherosclerosis and impairs lipid metabolism in mice, which may be mediated by F. nucleatum-promoted glycolysis and lipogenesis through PI3K/Akt/mTOR signalling in hepatocytes. Treatment of PD and specific targeting of F. nucleatum are promising strategies to improve therapeutic effectiveness of hyperlipidaemia and atherosclerosis.

Oral pathogens exacerbate Parkinson's disease by promoting Th1 cell infiltration in mice.

BACKGROUND: Parkinson's disease (PD) is a common chronic neurological disorder with a high risk of disability and no cure. Periodontitis is an infectious bacterial disease occurring in periodontal supporting tissues. Studies have shown that periodontitis is closely related to PD. However, direct evidence of the effect of periodontitis on PD is lacking. Here, we demonstrated that ligature-induced periodontitis with application of subgingival plaque (LIP-SP) exacerbated motor dysfunction, microglial activation, and dopaminergic neuron loss in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. RESULTS: The 16S rRNA gene sequencing revealed that LIP-SP induced oral and gut dysbiosis. Particularly, Veillonella parvula (V. parvula) and Streptococcus mutans (S. mutans) from oral ligatures were increased in the fecal samples of MPTP + LIP-SP treated mice. We further demonstrated that V. parvula and S. mutans played crucial roles in LIP-SP mediated exacerbation of motor dysfunction and neurodegeneration in PD mice. V. parvula and S. mutans caused microglial activation in the brain, as well as T helper 1 (Th1) cells infiltration in the brain, cervical lymph nodes, ileum and colon in PD mice. Moreover, we observed a protective effect of IFNγ neutralization on dopaminergic neurons in V. parvula- and S. mutans-treated PD mice. CONCLUSIONS: Our study demonstrates that oral pathogens V. parvula and S. mutans necessitate the existence of periodontitis to exacerbate motor dysfunction and neurodegeneration in MPTP-induced PD mice. The underlying mechanisms include alterations of oral and gut microbiota, along with immune activation in both brain and peripheral regions. Video Abstract.

Roles of oral microbiota and oral-gut microbial transmission in hypertension.

INTRODUCTION: Considerable evidence has linked periodontitis (PD) to hypertension (HTN), but the nature behind this connection is unclear. Dysbiosis of oral microbiota leading to PD is known to aggravate different systematic diseases, but the alteration of oral microbiota in HTN and their impacts on blood pressure (BP) remains to be discovered. OBJECTIVES: To characterize the alterations of oral and gut microbiota and their roles in HTN. METHODS: We performed a cross-sectional (95 HTN participants and 39 controls) and a 6-month follow-up study (52 HTN participants and 26 controls) to analyze the roles of oral and gut microbiota in HTN. Saliva, subgingival plaques, and feces were collected for 16S rRNA gene sequencing or metagenomic analysis. C57BL/6J mice were pretreated with antibiotics to deplete gut microbiota, and then transplanted with human saliva by gavage to test the impacts of abnormal oral-gut microbial transmission on HTN. RESULTS: BP in participants with PD was higher than no PD in both cross-sectional and follow-up cohort. Relative abundances of 14 salivary genera, 15 subgingival genera and 10 gut genera significantly altered in HTN and those of 7 salivary genera, 12 subgingival genera and 6 gut genera significantly correlated with BP. Sixteen species under 5 genera were identified as oral-gut transmitters, illustrating the presence of oral-gut microbial transmission in HTN. Veillonella was a frequent oral-gut transmitter stably enriched in HTN participants of both cross-sectional and follow-up cohorts. Saliva from HTN participants increased BP in hypertensive mice. Human saliva-derived Veillonella successfully colonized in mouse gut, more abundantly under HTN condition. CONCLUSIONS: PD and oral microbiota are strongly associated with HTN, likely through oral-gut transmission of microbes. Ectopic colonization of saliva-derived Veillonella in the gut may aggravate HTN. Therefore, precise manipulations of oral microbiota and/or oral-gut microbial transmission may be useful strategies for better prevention and treatment of HTN.

Endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway is involved in odontoblastic differentiation of human dental pulp cells.

OBJECTIVE: RNA-activated protein kinase-like ER-resident kinase (PERK) was a major transducer of Endoplasmic reticulum (ER) stress response and it directly phosphorylated α-subunit of eukaryotic initiation factor 2 (eIF2α), which specifically promoted the translation of activating transcription factor 4 (ATF4), an important transcription factor in cells' differentiation. The purpose of this study was to establish whether ER stress mediated by PERK-eIF2α-ATF4 pathway was involved in odontoblastic differentiation of human dental pulp cells (DPCs). METHODS: DPCs were isolated from extracted teeth and cultured in odontogenic medium. A recombinant lentiviral vector was constructed to transfect DPCs for PERK knockdown. Alkaline phosphatase (ALP) and Alizarin red S staining were used to characterize the odontoblastic differentiation. Real-time polymerase chain reactions (RT-PCR) were performed to analyze the genes' expressions in DPCs' odontoblastic differentiation. The mRNA and protein levels of ER stress markers were examined by RT-PCR and western blot. RESULTS: DPCs cultured in odontogenic media showed increased ALP activity and mineralized nodule formation. Notably, treatment with differentiation medium resulted in the up-regulation of genes, such as osteocalcin (OCN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), splicing x-box binding protein-1 (sXBP1), ATF4 and glucose-regulated protein 78 (GRP78). Meanwhile, the expressions of PERK-eIF2α-ATF4 pathway proteins, phosphorylated PERK, phosphorylated eIF2α and ATF4, increased in odontoblastic induction cells compared with controls. Furthermore, inhibition of PERK (PERK knockdown) decreased ALP activity and matrix mineralization in DPCs accompanied by the decrease expression of phosphorylated eIF2α and ATF4. CONCLUSION: These results suggested that PERK-eIF2α-ATF4 pathway was involved in the odontoblastic differentiation of DPCs.

[The risk and prevention of bio-aerosol transmission in dental clinics].

Corona virus disease 2019 (COVID-19) has become a Public Health Emergency of International Concern since its outbreak, and whether COVID-19 can transmit by aerosol remains controversial. The problem of bio-aerosol transmission in the relatively confined dental clinics has aroused wide attention in the field of dentistry. This review provided a most updated summary on the relation between bio-aerosols and dental clinics, which included the microorganisms in bio-aerosols, the bio-aerosol transmission and the sources testing methods, temporal and spatial distribution of dental bio-aerosols and summarized how to reduce the exposure to bio-aerosols in dental clinics.

[Management and prevention of dental emergency during corona virus disease 2019 (COVID-19) epidemic].

The outbreak of corona virus disease 2019 (COVID-19) has developed rapidly and the situation of prevention and control is severe. During the epidemic period of COVID-19, due to the particularity of diagnosis and treatment of oral diseases, there is great challenges for how to deal with various types of dental emergency. In order to prevent and control the epidemic situation strictly, and to perform a scientific and orderly clinical diagnosis and treatment of dental emergency, this article provided suggestions on personnel management training, procedures and treatment, protection and disinfection of dental emergency during COVID-19 epidemic, and reference for dental institutions and medical staff.

[Effect of recombinant connective tissue growth factor on proliferation and odontogenic differentiation of dental pulp cells].

PURPOSE: To analyze the effect of recombinant connective tissue growth factor(rCTGF) on proliferation and differentiation of human dental pulp cells(hDPCs). METHODS: Human dental pulp cells were cultured in vitro and treated with rCTGF at different concentrations (0, 1, 10, 100 ng/mL). The proliferation of dental pulp cells was detected by CCK8 assay. The formation of mineralized nodules was determined by alizarin red staining and half-quantitative alizarin Red S assay. qRT-PCR was utilized to detect the expression of odontogenic differentiation related genes DMP-1, DSPP and OC, and the phosphorylation level of ERK1/2 signaling pathway was detected by Western blot. The data were analyzed with SAS 9.3 software package. RESULTS: High concentration of rCTGF(100 ng/mL) could promote proliferation of dental pulp cells. After mineralization induction, 10 g/mL rCTGF had the best effect on promoting the formation of mineralized nodules in dental pulp cells, and calcium ion deposition was the most obvious(P<0.05). The expression of odontogenic differentiation related genes DMP-1 and DSPP was significantly up-regulated(P<0.05). Western blot results showed that hDPCs stimulated by 10 ng/mL rCTGF could increase the expression of p-ERK1/2. CONCLUSIONS: rCTGF may promote the proliferation and differentiation of human dental pulp cells through activating ERK1/2 signaling pathway.

[Effect of serum starvation and culture to confluence on cell cycle synchronization and mineralization of human dental pulp cells].

PURPOSE: To compare the effect of serum starvation and culture to confluence on cell cycle synchronization and mineralization of human dental pulp cells (hDPCs). METHODS: HDPCs were cultured to 80% and 100% confluence respectively, and then cultured for 24, 48 and 72 hours by culture medium containing 0.5% fetal bovine serum(FBS). Cell cycle of hDPCs were identified by flow cytometry. Then hDPCs cultured by serum starvation for 48h after culturing to 100% confluence were used as the experimental group, and hDPCs cultured to 80% confluence were used as the control group. The expression of alkaline phosphatase(ALP), collagen type Ⅰ(COL-Ⅰ) and osteocalcin(OCN) was detected at gene level; activity of ALPase was detected at protein level. SPSS 13.0 software was used for statistical analysis. RESULTS: When hDPCs were cultured by serum starvation for 48h after culturing to 100% confluence, cells at G0/G1 stage were more than culture to 100% confluence and serum starvation group (P＜0.05). At the genetic level, the expression of COL-Ⅰand OC in the experimental group was not statistically different from that of the control group, but can promote the expression of ALP(P＜0.05), and stimulate the secretion of hDPCs at protein level at the same time (P＜0.05). CONCLUSIONS: Culture to confluence combined serum starvation can synchronize more hDPCs at G0/G1 stage and promote mineralization of hDPCs.

Effect of Lactobacillus acidophilus and Porphyromonas gingivalis on proliferation and apoptosis of gingival epithelial cells.

PURPOSE: This study aimed to evaluate the possible antagonistic effects of Lactobacillus acidophilus on Porphyromonas gingivalis, and detect inhibition of Lactobacillus acidophilus on Porphyromonas gingivalis when they are co-cultured with human gingival epithelial cells. MATERIALS AND METHODS: Human gingival epithelial cells were co-cultured with Lactobacillus acidophilus and Porphyromonas gingivalis alone or together. The amount of Porphyromonas gingivalis adhering to or invading the epithelial cells were determined by bacterial counts. The cellular proliferation was assayed by the MTT method. Apoptosis was detected by flow cytometry with apoptosis detection kit. RESULTS: On one hand, Lactobacillus acidophilus reduced the inhibitory effect of Porphyromonas gingivalis on the human gingival epithelial cells proliferation in a dose dependent manner. On the other hand, Porphyromonas gingivalis induced significant apoptosis on human gingival epithelial cells, and Lactobacillus acidophilus inhibited this apoptosis-inducing effect of Porphyromonas gingivalis in a dose dependent manner. CONCLUSIONS: Porphyromonas gingivalis inhibits the proliferation and induces the apoptosis of human gingival epithelial cells. Lactobacillus acidophilus could attenuate this effect in a dose-dependent manner, and it thus reduces the destruction from pathogens. Lactobacillus acidophilus could be an effective candidate for probiotic therapy in periodontal diseases.

[Establishment of a model of endoplasmic reticulum stress response in dental pulp cells induced by tunicamycin].

PURPOSE: The aim of this study was to establish a model of endoplasmic reticulum (ER) stress in dental pulp cells(DPCs) induced by tunicamycin to better understand the molecular mechanism of DPCs related diseases mediated by ER stress. METHODS: DPCs were cultured using modified tissue explant technique in vitro and cultured in presence or absence of tunicamycin. DPCs' viability was measured by methylthiazol tetrazolium (MTT) assay. The mRNA level of ER stress markers was examined by RT-PCR. The data were analyzed with SPSS17.0 software package. RESULTS: The proliferative ability of DPCs decreased when exposed to tunicamycin in a dose-dependent manner. Treatment with tunicamycin resulted in up-regulation of ER stress genes, such as splicing x-box binding protein-1(sXBP1), activating transcription factor 4(ATF4), glucose-regulated protein 78(GRP78) and C/EBP homologous protein (CHOP). CONCLUSIONS: The results indicate that ER stress response is induced in DPCs by tunicamycin, and the ER stress model is successfully established.

[Elimination of Entercoccus faecalis with different disinfection methods in root canals in vitro].

PURPOSE: To compare the elimination effect against E.faecalis in root canals with different methods. METHODS: Fifty extracted premolars with single root canal were selected. After cleaning and autoclaving, they were contaminated by E.faecalis and incubated for 28 days as models. Then the models were randomly and equally divided into 5 groups and treated as below: specimens in group A were treated with saline irrigation, specimens in group B were treated with 3% NaClO irrigation (as positive control), specimens in group C were treated with PUI, specimens in group D were treated with diode laser radiation, specimens in group E were treated with combination of PUI and diode laser radiation. The specimens from root canals were collected by paper points. The bacterial suspensions were later serially diluted and plated on tryptic soy agar plates to enumerate the CFUs after 48 hours. Statistical analysis was performed using SAS 8.02 software package. RESULTS: As with all parts of the root canal in aggregate, the CFUs of the specimens treated with PUI, diode laser radiation and the combination of them were significantly lower than the specimens treated with saline irrigation (P<0.01), but there was no significant difference among 3 groups (P>0.05). However, the specimens treated with 3% NaClO irrigation had the best effect of disinfection. The number of CFUs in the specimens treated with 3% NaClO was almost zero. There was no significant difference between this group and others in CFUs(P<0.01). CONCLUSIONS: Specimens treated with PUI, diode laser radiation and the combination of them showed great effect of elimination against biofilm of Enterococcus faecalis compared with saline irrigation. Irrigation with 3% NaClO was the most efficient method in this experiment.

[Comparative evaluation of marginal microleakage of three different resins in Class V composite restorations].

PURPOSE: To compare and evaluate microleakage at the occlusal wall and cervical wall in Class V cavities restored with Ivoclar Tetric N Ceram Bulk Fill composite, Tetric N Flow composite and N Ceram nanocomposite. METHODS: Sixty-six extracted human maxillary premolars, which were intact and healthy, were randomly assigned into 3 groups (n=22). Standardized Class V cavities were prepared on the buccal surface of maxillary premolars. The occlusal walls of the cavities were located on the enamel and the cervical walls were located on dentin and cementum. After etching and application of the same bonding agents, the cavities were restored with different composite materials. Group A: Tetric N Ceram nano-hybrid composite , Group B: Tetric N Flow nano-hybrid composite, Group C: Tetric N Ceram Bulk Fill nano-hybrid composite. After curing with soft-start mode, finishing and polishing, the specimens were subjected to thermocycling. The specimens were coated with nail varnish, and immersed in 2% methylene blue solution for 7 days. The teeth were then sectioned longitudinally. Two of the samples chosen randomly from each group were evaluated under scanning electron microscope. Dye penetration of the remaining samples was examined with a stereomicroscope (×40) and scored separately for occlusal and gingival aspect on a 0-3 ordinal scale. The leakage depth was measured with Spot Advanced version 4.6 software package. The data were analyzed with Kruskal-Wallis, Mann-Whitney and Wilcoxon test using SPSS 17.0 software package. RESULTS: Tetric Bulk Fill had significantly less microleakage at the cervical margins than other groups (P<0.05). There was no significant difference at the occlusal margin (P>0.05) between the three groups. There was significant difference between the enamel and cervical wall microleakage (P<0.05). CONCLUSIONS: With the limitations of this study, Tetric Bulk Fill provided the least microleakage at the cervical wall among the three groups. There was no significant difference at the occlusal margin.

[Construction of a lentiviral vector of RNA interference of PERK gene and identification in human dental pulp cells].

PURPOSE: To construct an expression vector of a small hairpin RNA (shRNA) targeting human PERK gene and to observe gene-silencing effects of PERK in human dental pulp cells (DPCs). METHODS: According to PERK gene cDNA sequence, shRNA was designed and synthesized, which was then annealed into hU6-MCS-CMV-EGFP vector. After identified by sequencing, hU6-MCS-CMV-EGFP vector and packaging vector were co-transfected into 293 T cells. 72 hours later, the recombinant lentiviruses were obtained after harvesting and concentrating. Then LV-PERK-RNAi vectors were transfected into DPCs at an appropriate multiplicity of infection. To verify the interference effect, real- time PCR and Western blot were used to detect expression levels of PERK mRNA and protein in the transfected DPCs. The data were analyzed with SPSS 24.0 software package. RESULTS: LV-PERK-RNAi vectors were successfully constructed with a high titer of 3×108 TU/mL. The results of RT-PCR and Western blot demonstrated that after infection with LV-PERK-RNAi vector at a multiplicity of infection of 30, the expression level of PERK gene in DPCs was significantly down-regulated compared with control group. At mRNA level, the interference rate was about 63%. CONCLUSIONS: An effective lentiviral shRNA expression vector targeting the PERK gene is successfully constructed and can be used for further study on the function of PERK gene.

[Comparison of the accuracy of measuring root canal working length in 3 different ways].

PURPOSE: To compare the accuracy of measuring root canal working length with two different electronic apex locators and digital X-ray. METHODS: Ninety patients treated from January 2015 to June 2016 were selected, 232 root canals in total were randomly divided into 3 groups: X-ray group, Raypex5 group and i-root group. The accuracy of working length measured was assessed according to the filling quality of related main tips. The data were analyzed with SPSS19.0 software package for Chi-square test. RESULTS: The success rates for root canal filling of X-ray group, Raypex5 group and i-root group were 88.5%, 92.9% and 96.1% respectively with no significant difference between 3 groups (P>0.05). The success rates for root canal filling of molar teeth, premolar teeth and front teeth were 92.5%, 96.6% and 95.2% respectively with no significant difference between the 3 groups (P>0.05). CONCLUSIONS: The clinical effect of Raypex 5 and i-root electronic apex locators for working length measuring is similar to that of conventional X-ray, but the use of electronic apex locators can avoid unnecessary ionizing radiation and improve clinical efficiency.

[Evaluation of effects of fluoride varnish on prevention of first permanent molar caries].

PURPOSE: To compare fluoride varnish and fluoride foam's effectiveness of preventing first permanent molar caries, and discuss patients' acceptance of the 2 materials among pupil patients. METHODS: Six hundred pupils aged 6-8 years from 3 primary schools in Shanghai Yangpu District were randomly selected.The subjects were randomly divided into 3 groups, with 200 students having 800 teeth in each. Two of these groups were experimental groups that receive fluoride varnish (Group A) and fluoride foam (Group B) respectively, and the third group (group C) was control group.Pupils in group A and group B received 2.26% Toro fluoride fluoride varnish, 1.23% fluoride foam was applied to the first molars. In the next 2 years, all pupils received dental health education every half year. The pupils' conditions of first permanent molar caries were checked in the sixth, twelfth, eighteenth and twenty-fourth month since the study started. Statistical analysis was performed using SPSS 10.0 software package. RESULTS: After 6 months, there was no significant difference between the 3 groups (P>0.05), the rate of caries incidence in the experimental group B was significantly higher than group A during the observation period after 12 months (P<0.05). Caries incidence in both group A and B was lower than the control group, with significant difference (P<0.05). CONCLUSIONS: Both duraphat fluoride varnish and fluoride foam prove to be effective in caries prevention. Moreover, considering other factors such as safety, convenience and cost, fluoride protector will be an even better choice in practical use.

Differential expression between "DSP-only" and DSP-PP523 transcripts in rat molar teeth.

OBJECTIVE: To compare the expression patterns of two multiple transcripts derived from DSP-PP gene during tooth development. One is DSP-only transcript (i.e. does not encode PP) and the other is DSP-PP523 transcript, a main DSP-PP transcript. DESIGN: Unique antisense and sense riboprobes were generated from DSP-only and DSPPP523 cDNAs for in situ studies to examine DSP-only and DSP-PP523 transcript expression in developing molars. Paraffin-embedded sections (5-7µ m) from embryonic 20day, postnatal 2, 3 and 6days were deparaffined and hydrated. Tissues were prehybridized, then hybridized with DSP-only and DSP-PP523 anti-sense (AS) or sense (S) Digoxigenin labeled-riboprobes overnight, and washed. Anti-Digoxigenin antibodies conjugated to alkaline phosphatase were used to detect the presence of bound riboprobes by color reaction with NBT/BCIP. Stro-1 antibody was used for immunohistochemical analysis of Stro-1 protein expression in rat molars. RESULTS: We found that unlike the DSP-PP523 transcript, the DSP-only transcript does not express in the entire polarized mature odontoblasts but is expressed in the areas subjacent to the mature odontoblast layer. In addition, DSP-only transcript is expressed in the dental pulp. Interestingly, Stro-1 protein, a stem cell marker, was also identified in the areas subjacentto odontoblasts and in dental pulp. CONCLUSION: Differential expression of DSP-only and DSP-PP523 transcripts suggest that these two kinds of transcripts may play different roles during dentinogenesis. DSP-PP523 transcript is expressed in mature odontoblasts, which actively participates in dentin formation. DSP-only transcript might have a different function.

[Study of endoplasmic reticulum stress response in osteogenic differentiation of human periodontal ligament cells].

PURPOSE: The aim of this study was to establish whether endoplasmic reticulum (ER) stress is involved in osteogenic differentiation of periodontal ligament cells (PDLCs) and to better understand the mechanism of PDLCs osteogenic differentiation. METHODS: PDLCs were isolated from extracted teeth and cultured in presence or absence of osteogenic medium, which can induce osteogenic differentiation of PDLCs. Alkaline phosphatase and alizarin red S staining were performed to characterize the osteogenic differentiation of PDLCs. The mRNA and protein levels of ER stress markers were examined by RT-PCR and Western blot analysis. The data were analyzed with SPSS 17.0 software package. RESULTS: Cell treated with osteogenic medium showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation compared with untreated controls. Treatment with osteogenic induction resulted in up-regulation of genes, such as splicing x-box binding protein-1 (sXBP1), activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78). The expressions of ER stress protein markers, phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), increased in osteogenic induction cells compared with controls. CONCLUSIONS: The results indicate that ER stress response is involved in the process of osteogenic differentiation of PDLCs.

[Clinical management of calcified root canal with Mtwo NiTi files].

PURPOSE: To evaluate the effect of Mtwo NiTi files in the management of calcified root canals. METHODS: Thirty-five calcified root canals from 21 cases unsuccessfully prepared with C-Pilot files and EDTA were treated with Mtwo NiTi files and EDTA. The data was analyzed with SAS 9.4 software package. RESULTS: Twenty-three calcified root canals were successfully prepared. The success rate was 65.71%. Instrument separation occurred in 4 root canals, and ledge occurred in 3 root canals. Categorized by calcified position, the success rate of three groups had no significant differences (P>0.05). CONCLUSION: Mtwo NiTi files can effectively process calcified root canals.