[Single-cell transcriptome analysis reveals development atlas of mouse molar pulp cells].

Objective: Single-cell RNA sequencing (scRNA-seq) was used to analyze the developing mouse molars, in order to construct a spatiotemporal development atlas of pulp cells, and further to reveal the developmental process and regulatory mechanism of tooth development. Methods: Ten mandibular first molars from C57BL/6 mice in postnatal day (PN) 0 and 3 were respectively dissected and digested to obtain single-cell suspensions. scRNA-seq was performed on 10× Genomics platform. PN 7 mouse molar scRNA-seq data were obtained from our previous study. PN 0, 3, and 7 scRNA-seq data were integrated for following analysis. The initial quality control, mapping and single cell expression matrix construction were performed by Cell Ranger. Quality control, standardization, dimensional reduction and cluster analysis were performed by using Seurat. Monocle was used to generate the pseudotime trajectory. Scillus was used to perform gene ontology analysis. In order to detect the spatiotemporal change of different population of pulp cells, the marker genes of each cluster were demonstrated by RNAscope in situ hybridization. Results: There were twenty-six cell clusters within mouse molars, which were identified as eight different cell types, including dental pulp cells, dental follicle cells, epithelial cells, immune cells, endothelial cells, perivascular cells, glial cells and erythrocytes. We further re-clustered and analyzed dental pulp cells. Cluster 0 were mature pulp cells, which located at the upper portion of crown. The main functions of cluster 0 were osteogenesis and extracellular structure organization. Cluster 1 were apical papilla cells, which located at the apical part of roots, whose main functions were extracellular structure organization and organ development. Cluster 2 were cycling cells, which were actively proliferated, resided in the lower portion of the crown. Cluster 3 and 4 were preodontoblasts and odontoblasts, respectively. Their functions were closely related to biomineralization. The proportion of mature pulp cells increased with the development process, while the proportion of cycling cells and odontoblast lineage decreased. According to the expression pattern of marker genes of each cluster, we constructed a cell atlas of dental pulp. Pseudotime trajectory analysis found there were two development trajectories within dental pulp. They both started from SPARC related modular calcium binding 2 (Smoc2)+ dental papilla cells, then went through DNA topoisomerase Ⅱ alpha (Top2a)+ cycling cells, and finally divided into coxsackie virus and adenovirus receptor (Cxadr)+ mature pulp cells or dentin sialophosphoprotein (Dspp)+ odontoblasts two lineages. Conclusions: scRNA-seq could fully discover the intercellular heterogeneity of cells on transcriptome level, which provides a powerful tool to study the process and regulatory mechanism of organ development.

[Preliminary study on the relationship between different blood glucose levels and periodontitis in patients with diabetes mellitus type 2].

Objective: To determine the correlation between the diabetes mellitus control and periodontitis. Methods: This study was a cross-sectional survey using stratified system sampling model design. The target population was the patients with diabetes investigated from May to July 2018 in Huangpu District of Shanghai. In the present study, severe periodontitis was defined as at least at two sites in different quadrants with probing depth (PD)≥6 mm and clinical attachment loss (CAL)≥ 5 mm. Edentulous induced by periodontitis were also classified as severe periodontitis and the others were classified as non-severe periodontitis subjects. Diabetes control levels were divided into the following three groups: poorly controlled group [glycated hemoglobin (HbA1c)>7.5% and fasting blood glucose (FPG)>7.0 mmol/L], well controlled group (6.5%≤HbA1c≤7.5％ or 6.1 mmol/L≤FPG≤7.0 mmol/L) and ideally controlled group (HbA1c<6.5% and FPG<6.1 mmol/L). SPSS 25.0 was used for statistical analysis. Chi square test was used for demographic data and frequency distribution, α=0.05, two-sided test. Ordinal regression model was used for PD and diabetes control status to balance confounding factors (including age, gender, education and smoking status). After matching the propensity scores between severe periodontitis group and non-severe periodontitis group, logistic regression analysis was used to analyze the level of diabetes control and periodontitis. Results: A total of 5 220 adults over the age of 18 with a medical history of diabetes participated in the survey, of which 3 064 subjects with diabetes mellitus type 2 (T2DM) who were given both oral and laboratory examinations and were included in this study. Statistics showed that the prevalence of moderate and severe periodontitis was 10.57% (324/3 064). In the severe periodontitis group, 79.01% (256/324) of the subjects were over 65 years old, 55.56% (180/324) were male, 58.33% (189/324) had lower education level than high school level, and 21.91% (71/324) were smokers, which were significantly higher than those in the non-severe periodontitis group (P<0.01). In different T2DM status groups, the percentage of severe periodontitis increased with the aggravation of T2DM status. In severe periodontitis group, the proportion of patients with poor glycemic control was higher. T2DM patients with poor glycemic control accounted for 68.52% (222/324) in severe periodontitis group, which was significantly higher than the proportion of non-severe periodontitis group of 60.99% (1 671/2 740) (P<0.05). The regression coefficient of PD was 0.191, and PD had a significant negative effect on the level of blood glucose (P<0.01). There was a significant positive correlation between diabetes glycemic control and severe periodontitis (OR=2.800, P<0.05). Conclusions: In Huangpu District of Shanghai, among T2DM patients, the age of severe periodontitis group was higher than that of non-severe periodontitis group, most of them were male, with lower education level and higher proportion of smoking. The severity of diabetes was related to periodontitis and the proportion of severe periodontitis was higher in patients with poor glycemic control.

[Analysis of caries experience and the dental treatments under general anesthesia in 103 cases of children with autism spectrum disorders].

Objective: To compare the caries experience and the kinds of dental treatment between children with autism spectrum disorders (ASD) and children without systemic disease who were all treated under general anesthesia. Methods: Totally 103 children with ASD who received dental treatments under general anesthesia in 13 professional dental hospitals around China from April to November 2016 were included in the present study. A group of 97 children without systemic disease, according to the age, gender and application propensity score matching method, were chosen as controls, who received dental treatments under general anesthesia between January 2015 to November 2018 in the same hospitals as the children with ASD. Decay missing filling tooth (DMFT/dmft, DMFT for permanent teeth and dmft for primary teeth) indices of two groups of children and the contents of the dental treatments under general anesthesia were analyzed. Results: No significant difference of DMFT/dmft index ï¼»M (Q 25, Q 75)ï¼½ was found between children with ASD group ï¼»0 (0, 3)/11(8, 14)ï¼½ and control group ï¼»0 (0, 3)/9(7, 13)ï¼½ (P>0.05). The average number of dental treatments under general anesthesia and the average number of endodontic treatment in children with ASD were 13 (11, 15) and 3 (2, 6) teeth respectively, while those in the control group were 12 (9, 14) and 2 (1, 4) teeth respectively, the differences were statistically significant (P<0.01, P<0.05). Conclusions: No significant difference was found between children with ASD and the normal controls who receive dental treatments under general anesthesia in DMFT/dmft index, but the treatment needs of children with ASD is relatively higher, and their tooth decay is relatively severer.

[Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells].

OBJECTIVE: To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. METHODS: SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. RESULTS: The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 µm) and SHED (7 734.3 µm) was higher than DPSC (5 541.0 µm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). CONCLUSION: SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

[Effect of exogenous stem cells from apical papillae in the pulp revascularization treatment for the immature permanent tooth with periapical periodontitis].

Objective: To evaluate the effect of exogenous stem cells from apical papillae (SCAP) in the pulp revascularization treatment for the immature permanent tooth with periapical periodontitis in animal model. Methods: After the SCAP were isolated and cultured from the Beagle dogs, stem cell properties of these cells were characterized by analyzing their colony-forming ability, the expression of mesenchymal stem cell markers and the multidifferentiation characteristics including osteogenic, adipogenic, and chondrogenic potentials. Models of young permanent tooth with periapical periodontitis were established in dogs and the infection in each of the model tooth was eliminated by root canal irrigation and intracanal medication. After that, all of the model teeth were randomly divided into 4 groups: Group 1: normal developing teeth with no treatment applied;Group 2: teeth that periapical tissues were irritated to induce blood flowing into the root canals;Group 3: teeth that peripheral blood was delivered into the root canals;Group 4: teeth that SCAP were resuspended in peripheral blood and delivered into the root canals. In Group 2-4, firm coronal seal was performed after revascularization procedure and radiographs were taken periodically in order to observe the development of roots. After a 12-week-period, alveolar samples were collected and observed histologically. Results: The isolated SCAP showed clonogenic ability and multilineage differentiation ability including osteogenic, adipogenic and chondrogenic potentials. These cells also expressed the mesenchymal stem cell markers such as STRO-1 and CD146, while no cytokeratin was detected. The thickening of canal wall was observed radiographically 12 weeks after procedures of infection control and revascularization. Histologically, the newly formed tissues on the inner canal wall were found bone lacuna like structure in Group 2 and 3, and the new tissue formed in the Group 3 seemed easy to separate from the canal wall. The newly formed tissues in Group 4 were much thicker compare to those in the Group 2 and 3, and the dentine tubule like structure instead of bone lacuna was noticed although the orientation of these tubules were various. Conclusions: SCAP seem to play an important role in the tissue regeneration procedure when infection is well controlled in young permanent teeth with periapical periodontitis. It is difficult to achieve real tissue regeneration due to the lack of endogenous SCAP in apical area, therefore delivering adequate exogenous SCAP isolated and cultured in vitro could be a promising approach to overcome the challenge.

[Three-dimensional analysis of the early correction of anterior crossbite using eruption guidance appliance].

OBJECTIVE: To investigate the clinical effect of eruption guidance appliance in the treatment of anterior cross bite in mixed-dentition children. METHODS: In the study,10 mixed-dentition children with anterior cross bite, totally 12 incisors, were selected. Alginate was used to take upper and lower dentition impression and make a hard plaster model,which served as the eruption guidance appliance for treatment. The pre- and post-operative dental casts were digitized with SmartOptics Activity 880 scanner,and the three-dimensional overlapping models were obtained by reverse engineering software,Geomagic Studio 2012,then the three-dimensional movements of the upper and lower incisors were analyzed using Imageware 13.2 software. The overbite and overjet were analyzed using the same methods. Measurement with copper wire was used to analyze the upper and lower arch length. Space analysis was the result by the sum of crown width minus the arch length. The crown width of unerupted permanent teeth was according to X-ray method to predict. The SPSS 17.0 software was used to analyze the pre- and post-operative measurements of the same child. The normality test of the measured data showed that it conformed to the normal distribution. Therefore,the t test and double side test were used,and the significance level was 0.05. RESULTS: The course of treatment was (5.6±2.7) months. During orthodontic treatment, the upper incisors moved mainly labially (P<0.001) in three-dimensional displacement, and the lower incisors moved mainly the same direction (P=0.025). During the treatment of eruption guidance appliance,the average overbite decreased (1.01±0.9) mm (t=-3.531, P=0.006), and the difference was statistically significant. There was no statistically significant difference between the pre- and post-operative average overjet (t=0.771, P=0.460). The severity of crowding in upper arch decreased (1.9±0.99) mm (t=-6.042, P<0.001),and that in lower arch decreased (1.9±0.74) mm (t=-8.143, P<0.001), both of the differences were statistically significant. CONCLUSION: The anterior cross bite in mixed dentition could be corrected by eruption guidance appliance, and at the same time, the normal overjet and overbite were established, and the teeth were aligned.

[Comparison of the properties of CD146 positive and CD146 negative subpopulations of stem cells from human exfoliated deciduous teeth].

OBJECTIVE: Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering. METHODS: In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR. RESULTS: SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation. CONCLUSION: The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.

[Recombinant human transforming growth factor ß1 promotes dental pulp stem cells proliferation and mineralization].

OBJECTIVE: To explore suitable concentration of recombinant human transforming growth factor ß1 (rhTGF-ß1) usage and study the effect of rhTGF-ß1 on differentiation of dental pulp stem cells (DPSCs). METHODS: DPSCs were isolated from the undiseased third molars of people aged 18-25 years and cultured according to instructions in vitro. Different concentrations (1 , 6 , 10 µg/L) of rhTGF-ß1 were added to the culture medium to examine DPSCs proliferation by CCK-8 (cell counting kit-8) assay.The suitable concentration was then selected. For differentiation, the DPSCs were incubated for 7 or 14 days with rhTGF-ß1 supplemented with osteo/odontoblastic induction medium containing 10 nmol/L dexamethasone, 10 mmol/L b-glycerophosphate, 50 g/L ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvitamin D3 and 10% fetal bovine serum. The cells were then washed 3 times with phosphate-buffered saline and sonicated with 1%Triton X-100 for 30 minutes on ice. Cellular alkaline phosphatase (ALP) activity was assayed with p-nitrophenyl phosphate as the substrate. The enzyme activity was expressed as p-nitrophenyl produced per milligram of protein [bicinchoninic acid (BCA) protein assay kit]. To examine mineral nodule formation, the cultured cells were fixed in 4% paraformaldehyde and washed in water, and the mineralization of the extracellular matrix was assayed by 1% alizarin red S staining and elution of staining was examined as optical density (D) under microplate reader. The mean difference was considered significant at 0.05 and 95% confidence interval. RESULTS: The DPSCs had typical fibroblast morphology and could form mineral nodules after being cultured with osteo/odontoblstic induction medium for 14 days. 6 µg/L rhTGF-ß1 significantly promoted the DPSCs proliferation on the 3rd and 5th days. After the incubation of osteo/odontoblastic induction medium, the DPSCs with the 6 µg/L rhTGF-ß1 increased ALP activities compared with the control; D values in the 6 µg/L rhTGF-ß1 group was 0.31±0.03, while the control group was 0.02±0.01 (P<0.05). The total protein content in the 6 µg/L rhTGF-ß1 group was (2 775.46±83.54) mg/L, and the control group was (1 432.20±110.83) mg/L (P<0.05). To eliminate the cells proliferation influence, relative ALP activities, which was defined as the total ALP divided by the total protein content, the 6µg/L rhTGF-ß1 group was 6 times higher than the control group. Alizarin red S staining showed increased mineral nodule formation in the rhTGF-ß1 group. The elution of staining under microplate reader also showed more optical density in the 6 µg/L rhTGF-ß1-treated cells (0.83±0.02) than that in the control groups (0.55±0.05, P<0.05). CONCLUSION: 6 µg/L rhTGF-ß1 could significantly promote DPSCs proliferation and odontoblastic differentiation in vitro.

[Comparison of long-term dental treatment effects of children treated under general anesthesia and passive restraint].

Objective: To compare the long-term dental treatment effects, oral health habits and oral-health-related qualities of life of children treated under general anesthesia (GA) and passive restraint (PR), respectively. Methods: Twenty seven 2 to 4-year-old children treated under GA and thirty four children treated under PR were recruited in the Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology. Up to 2 years after the treatment, a follow up assessment was conducted. The data of general information, dental plaque level and the unplanned treatments were recorded and analyzed. The questionnaire of oral health habits and early childhood oral health impact scale (ECOHIS) for each child was also completed and analyzed. The survival rate and median survival time of the deciduous teeth were calculated. Multivariate analysis was performed by using Cox proportional hazard model. Results: Twenty-five children under GA and 32 under PR were finally included, with a total of 1 098 deciduous teeth. The postoperative dental plaque indicesin both GA and PR groups had significantly improved than that of before the treatments (P=0.019, P<0.001). The oral health habits had also improved, and the improvement in PR group was more obvious than that in GA group. Totally 128 teeth (27.0%) appeared unplanned treatments in GA group and 232 teeth (37.2%) in PR group during the follow-ups. The new caries and recurrent caries in PR group were significantly more than that in GA group (P<0.001, P=0.012). No significant differences were found between the two groups in restoration failure, secondary caries and endodontic diseases (P=0.129, P=0.822, P=0.642). However, the time of occurrence of endodontic disease and secondary caries in GA group were significantly longer than that in PR group (P<0.01, P<0.001). The median survival time of teeth in GA group was 1 018 days comparing to 944 days in PR group. The difference was statistically significant (P<0.05). The survival rate was associated with such factors as decayed-missing-filled tooth (dmft), anterior or posterior teeth, feeding frequency, brushing habits and behavior management techniques. Conclusions: The long-term dental treatment effects of children treated under GA was significantly better than that of PR group. Continuous reinforcement of proper dietary and oral hygiene habits might help in maintaining the long-term treatment effect.

[A 2-year cohort study on the caries risk assessment of 3-year-old caries-free children using Cariostat caries activity test].

Objective: To evaluate the caries status of a cohort of 3-year-old caries-free children from 2 kindergartens in Beijing in a period of 2 years by using Cariostat caries activity test and to assess the sensitivity and specificity of Cariostat score as a caries risk indicator for caries-free children. Methods: Totally 426 3-year-old caries-free children from 2 kindergartens in Beijing were recruited in the present study. Informed consents were obtained from the children's parents. Dental plaque samples of the children were collected and the Cariostat caries activity tests were conducted at baseline and once a year for 2 years. After two years, the caries status of the cohort children were re-evaluated and the caries incidences amongst children with high (2.0, 2.5, 3.0), medium (1.5) and low (1.0, 0.5, 0.0) levels of Cariostat scores were compared and analyzed. Results: Totally 864 3-year-old children from 2 kindergartens were screened before the study startedand 426 (49.3%) children were caries free. After 2-year follow-up, 312 out of 426 (73.2%) remained in the study. The overall caries incident rate was 46.5% (145/312). The caries incident rate of children with high level of Cariostat scores was 88.9% (88/99), while the caries incident rates of children with medium and low levels of Cariostat scores was 38.7% (36/93) and 17.5% (21/120), respectively. The sensitivity and specificity of the Cariostat test in assessing the caries risk of 3-year-old caries-free children in a period of 2 years were 60.7% and 93.4%, respectively. Conclusions: Cariostat caries activity test can be used as an indicator to predict the caries risk of 3-year-old caries-free children. Comprehensive caries management could be conducted for children in kindergartens based on the caries risk assessment results of caries experience and the Cariostat score.

[Effects of breast-feeding duration, bottle-feeding duration and oral habits on the occlusal characteristics of primary dentition].

OBJECTIVE: To assess the effects of breast-feeding duration, bottle-feeding duration and oral habits on the occlusal characteristics of primary dentition in 3-6-year-old children in Beijing. METHODS: This cross sectional study was conducted via an examination of the occlusal characteristics of 734 children combined with a questionnaire completed by their parents/guardians. The examination was performed by a single, previously calibrated examiner and the following variables were evaluated: presence or absence of deep overbite, open bite, anterior cross bite, posterior cross bite, deep overjet, terminal plane relationship of the second primary molar, primary canine relationship, crowding and spacing. Univariate analysis and multiple Logistic regressions were applied to analyze the associations. RESULTS: It was found that a short duration of breast-feeding (never or ≤6 months) was directly associated with posterior cross bite (OR=3.13, 95%CI=1.11-8.82, P=0.031) and no maxillary space (OR=1.63, 95%CI=1.23-2.98, P=0.038). In children breast-fed for ≤6 months, the probability of developing pacifier-sucking habits was 4 times that for those breast-fed for >6 months (OR=4.21, 95%CI=1.85-9.60, P=0.000 2). The children who were bottle-fed for over 18 months had a 1.45-fold higher risk of nonmesial step occlusion and a 1.43-fold higher risk of class II canine relationship compared with those who were bottle-fed for 6-18 months. Non-nutritive sucking habits were also found to affect occlusion: a prolonged digit-sucking habit increased the probability of an anterior open bite, while a pacifier-sucking habit was associated with excessive overjet and absence of lower arch developmental space. Tongue-thrust habit was associated with anterior open bite (OR=4.21, 95%CI=1.85-9.60, P=0.000 2) and posterior cross bite (OR=7.24, 95%CI=1.30-40.13, P=0.024). Lower lip sucking habit was associated with deep overjet and had a negative association with class III canine relationship. Unilateral chewing was associated with spacing in mandibular (OR=1.57, 95%CI=1.03-2.41, P=0.037). Mouth breathing was associated with chronic rhinitis and adenoidal hypertrophy and had an association with spacing in maxillary. The chi-square test did not indicate a statistically significant association between upper lip sucking habit and any occlusal characteristics. CONCLUSION: Breast-feeding duration was shown to be associated with the prevalence of posterior crossbite, or no maxillary space in the deciduous dentition and development of a pacifier-sucking habit. Children who had a longer duration of bottle-feeding were more likely to develop class II canine relationship. Children who had an oral habit were more likely to develop abnormal occlusal characteristics.

[Impact of different degree pulpitis on cell proliferation and osteoblastic differentiation of dental pulp stem cell in Beagle immature premolars].

OBJECTIVE: To compare the proliferation and osteoblastic differentiation of dental pulp stem cell (DPSC) isolated from normal and inflamed pulps of different degrees in Beagle immature premolars, and provide evidence for the use of inflammatory DPSC (IDPSC). METHODS: This study evaluated 14 Beagle's young premolars (21 roots). In the experiment group, irreversible pulpitis was induced by pulp exposure and the inflamed pulps were extracted 2 weeks and 6 weeks after the pulp chamber opening.For the control group, normal pulps were extracted immediately after the exposure. HE staining and real-time PCR were performed to confirm the inflammation. The cells were isolated from the inflamed and normal pulps (IDPSC and DPSC). Cell proliferation and osteoblastic differentiation potentials of the two cells were compared. RESULTS: Inflammation cells infiltration was observed in the inflamed pulps by HE staining. The expression of inflammatory factor was much higher in the 6 week inflamed pulp. IDPSC had higher potential of cell proliferation and osteoblastic differentiation potentials. Furthermore, the osteoblastic differentiation potentials of IDPSC from 2 week inflamed pulp were higher than those from 6 week inflamed pulp. CONCLUSION: The potential of cell proliferation and osteoblastic differentiation of DPSC was enhanced at early stage of irreversible pulpitis, and reduced at late stage in Beagle immature premolars.

RUNX2 Mutation Impairs 1α,25-Dihydroxyvitamin D3 mediated Osteoclastogenesis in Dental Follicle Cells.

Cleidocranial dysplasia (CCD), a skeletal disorder characterized by delayed permanent tooth eruption and other dental abnormalities, is caused by heterozygous RUNX2 mutations. As an osteoblast-specific transcription factor, RUNX2 plays a role in bone remodeling, tooth formation and tooth eruption. To investigate the crosstalk between RUNX2 and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) in human dental follicle cells (hDFCs) during osteoclast formation, we established a co-culture system of hDFCs from CCD patient and healthy donors with peripheral blood mononuclear cells (PBMCs). Expression of the osteoclast-associated genes and the number of TRAP(+) cells were reduced in CCD hDFCs, indicating its suppressed osteoclast-inductive ability, which was reflected by the downregulated RANKL/OPG ratio. In addition, 1α,25-(OH)2D3-stimulation elevated the expression of osteoclast-related genes, as well as RANKL mRNA levels and RANKL/OPG ratios in control hDFCs. Conversely, RUNX2 mutation abolished this 1α,25-(OH)2D3-induced RANKL gene activation and osteoclast formation in CCD hDFCs. Therefore, RUNX2 haploinsufficiency impairs dental follicle-induced osteoclast formation capacity through RANKL/OPG signaling, which may be partially responsible for delayed permanent tooth eruption in CCD patients. Furthermore, this abnormality was not rescued by 1α,25-(OH)2D3 application because 1α,25-(OH)2D3-induced RANKL activation in hDFCs is mediated principally via the RUNX2-dependent pathway.

[Children's intelligence quotient following general anesthesia for dental care: a clinical observation by Chinese Wechsler young children scale of intelligence].

OBJECTIVE: It has been demonstrated that anesthetics exposure may lead to neurocognitive impairment in developing brain of animal models. However, for the limitation that the animal models cannot fully mimic the dose and duration in clinical settings especially for dental general anesthesia, the clinical significance of anesthetics exposure on developing central nervous system remains undetermined. Therefore, we conducted the current study in order to observe the fluctuation of intelligence quotient (IQ) after the administration of dental general anesthesia comparing to that before surgery. We conducted the current study in order to observe the fluctuation of intelligence quotient (IQ) after the administration of dental general anesthesia compared with that before surgery. METHODS: Thirty two patients, ASA I, who were exposed to dental general anesthesia in Department of Pediatric Dentistry Peking University School and Hospital of Stomatology, aged 4 to 6.5 years, were enrolled in this prospective study. Patients with severe learning difficulties or communication disorders were excluded. Written and informed consent was obtained from each patients' family which was fully explained of the purpose and method of study. Their intelligence quotients were evaluated with the Chinese Wechsler young children scale of intelligence (Urban version) before and 2 weeks after dental anesthesia. They were treated by experienced pediatric dentists and the sevoflurane, propofol and nitrous oxide were used for general anesthesia by anesthetist. Articaine hydrochloride and epinephrine tartrate injections were used for their pulp treatment or extraction. The examiners and scorers for IQ had technical training in the test administration. All the patients were tested by the same examiner and with standardized guide language. Each subtest was scored according to the tool review. Verbal IQ and performance IQ consisted of relevant 5 subtests and full scale IQ. Statistical analyses were performed by SPSS 18.0. All the scores of subtests and 3 types of IQ were expressed as mean±standard deviation. Paired two-tailed t test was applied and P<0.05 was accepted as statistically significant. RESULTS: The results of intelligent assessment from 28 subjects were collected. The anesthetic time was (163.4±32.6) min and the number of treated teeth was 12.1±2.3, mean age (4.60±0.41) years; age range=4.04 to 5.44 years. Among the patients, there were 13 girls and 15 boys. There was no significant difference in gender distribution. The postoperative full IQ (128.46±10.85) was higher than that before surgery (124.64±11.46, P= 0.017). We found that the elevation of performance IQ, to a large extent, contributed to this change in full IQ (P= 0.007). Correspondingly, there was no statistical difference in the verbal IQ, which was 119.68±11.74 to 120.21±15.61 (P=0.854). CONCLUSION: Dental general anesthesia has no negative effect on the intelligence of preschool children, who were treated under general anesthesia by sevoflurane, propofol and nitrous oxide for 2 to 4 hours. The full IQ and performance IQ were slightly enhanced after treatment which can be explained by the memory effect.

Predictive role of RRM1 and BRCA1 mRNA expression on the clinical outcome of advanced non-small cell lung cancer.

This study aimed to evaluate the association between RRM1 and BRCA1 expressions and the therapeutic efficacy of platinum-based chemotherapy in non-small cell lung cancer patients in terms of their response and prognosis. In total, 377 patients agreed to participate in our study, and all of them received platinum-based combination chemotherapy between January 2008 and January 2009. The relative cDNA quantitation for RRM1 and BRCA1 was conducted using a fluorescence-based, real-time detection method, using ß-actin as a reference gene. The average age of the 377 patients was 64.6 years (range: 25.5-86.4 years), including 269 men and 108 women. Patients with high RRM1 expression benefited more from a platinum-containing regimen, and patients with high BRCA1 expression showed a high response rate to a platinum-containing regimen and reduced disease progression. Patients with high RRM1 expression were associated with a longer progression-free survival (PFS) and overall survival (OS) than those with low expression, and the hazard ratios (HRs) (95% confidence interval (CI)) were 0.67 (0.32-0.91) and 0.54 (0.30-0.95), respectively. Patients with high BRCA1 expression showed longer PFS and OS compared to those with low expression, and the HRs (95%CI) were 0.54 (0.30-0.95) and 0.62 (0.32-0.93), respectively. These results could be used in personalized chemotherapy decisions and to increase the response rate and prolonged survival, and could encourage exploration of the predictive value of other genes.

Novel RUNX2 mutations in Chinese individuals with cleidocranial dysplasia.

UNLABELLED: Cleidocranial dysplasia (CCD) is an inherited autosomal-dominant skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor, RUNX2. We performed mutation analysis of RUNX2 on four unrelated Chinese individuals with CCD. Three novel distinct mutations were detected in the coding region of RUNX2: two missense and one frameshift. These mutations were exclusively clustered within the Runt domain. One missense mutation converts threonine to isoleucine at codon 200 (T200I). The other one substitutes leucine for arginine at codon 225 (R225L), which affects many family members. The frame-shift mutation (214fs) in exon3 leads to the introduction of a translational stop codon at codon 221, resulting in a truncated RUNX2 protein. The reporter gene assays revealed that all the mutants exhibited significantly reduced transactivation activities on the osteocalcin promoter. Our results provide new genetic evidence that mutations involved in RUNX2 contribute to CCD. ABBREVIATIONS: AML3, gene encoding acute myeloid leukemia protein 3; bp, base pair; CBFA1, gene encoding core-binding factor 1; CBFbeta, gene encoding core-binding factor beta; CCD, cleidocranial dysplasia; NLS, nuclear localization signal; OSE2, osteoblast-specific cis-acting element 2; PEBP2A, gene encoding polyoma enhancer binding protein 2A; PST, proline/serine/ threonine-rich domain; Q/A, glutamine-alanine repeat domain; Runt, Runt Homology Domain; RUNX2, the mammalian runt-related genes 2; RUNX2, Runt-related protein 2.

Initial comparison of proteomic profiles of whole unstimulated saliva obtained from generalized aggressive periodontitis patients and healthy control subjects.

BACKGROUND AND OBJECTIVE: Salivary proteomics technology can be used to evaluate the disease progression of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups. MATERIAL AND METHODS: Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry. RESULTS: Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) gamma2 chain C region, Ig alpha2 chain C region, vitamin D-binding protein, salivary alpha-amylase and zinc-alpha2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased. CONCLUSION: Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.

Glycine receptors are functionally expressed on bullfrog retinal cone photoreceptors.

Using immunocytochemical and whole cell recording techniques, we examined expression of glycine receptors on bullfrog retinal cone photoreceptors. Immunofluorescence double labeling experiments conducted on retinal sections and isolated cell preparations showed that terminals and inner segments of cones were immunoreactive to both alpha1 and beta subunits of glycine receptors. Moreover, application of glycine induced a sustained inward current from isolated cones, which increased in amplitude in a dose-dependent manner, with an EC50 (concentration of glycine producing half-maximal response) of 67.3+/-4.9 microM, and the current was blocked by the glycine receptor antagonist strychnine, but not 5,7-dichlorokynurenic acid (DCKA) of 200 microM, a blocker of the glycine recognition site at the N-methyl-D-aspartate (NMDA) receptor. The glycine-induced current reversed in polarity at a potential close to the calculated chloride equilibrium potential, and the reversal potential was changed as a function of the extracellular chloride concentration. These results suggest that strychnine-sensitive glycine receptors are functionally expressed in bullfrog cones, which may mediate signal feedback from glycinergic interplexiform cells to cones in the outer retina.

[A study of fluoride and dexamethasone affecting the expression of growth factors in osteoblast].

OBJECTIVES: Investigate the expression of growth factors or receptors in osteoblast, to understand the possible mechanism of low dose fluoride and dexamethasone on the development and differentiation of osteoblast. METHODS: Investigate the expression of platelet derived growth factors (PDGF-A) and insulin-like growth factors (IGF-I-Ralpha) in osteoblast cultured in vitro by immunohistochemistry, furthermore, to deal with the results by image analysis. RESULTS: There were no significant effects of PDGF-A both of low doses fluoride and dexamethasone treated on the MC3T3-E1 osteoblast during the various stages of the development. Otherwise, the expression of IGF-I-Ralpha might increase treated with them at the early stage of the cell culture, and dexamethasone did more significantly. CONCLUSION: Low doses of fluoride or dexamethasone may promote the expression of IGF-I-Ralpha in osteoblast precursors, which maybe contribute to the effects of fluoride and dexamethasone on the proliferation and differentiation of osteoblast.