Evolutionary and Pan-genome Analysis of Three Important Black-pigmented Periodontal Pathogens.

OBJECTIVE: To analyse the pan-genome of three black-pigmented periodontal pathogens: Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens. METHODS: Pan-genome analyses of 66, 33 and 5 publicly available whole-genome sequences of P. gingivalis, P. intermedia and P. nigrescens, respectively, were performed using Pan-genome Analysis Pipeline software (version 1.2.1; Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China). Phylogenetic trees were constructed based on the entire pan-genome and single nucleotide polymorphisms within the core genome. The distribution and abundance of virulence genes in the core and dispensable genomes were also compared in the three species. RESULTS: All three species possess an open pan-genome. The core genome of P. gingivalis, P. intermedia and P. nigrescens included 1001, 1514 and 1745 orthologous groups, respectively, which were mainly related to basic cellular functions such as metabolism. The dispensable genome of P. gingivalis, P. intermedia and P. nigrescens was composed of 2814, 2689 and 906 orthologous groups, respectively, and it was enriched in genes involved in pathogenicity or with unknown functions. Phylogenetic trees presented a clear separation of P. gingivalis, P. intermedia and P. nigrescens, verifying the reclassification of the black-pigmented species. Furthermore, the three species shared almost the same virulence factors involved in adhesion, proteolysis and evasion of host defences. Some of these virulence genes were conserved across species whereas others belonged to the dispensable genome, which might be acquired through horizontal gene transfer. CONCLUSION: This study highlighted the usefulness of pan-genome analysis to infer evolutionary cues for black-pigmented species, indicating their homology and phylogenomic diversity.

Changes of Microbial Community in Treated Peri-implantitis Sites: An Experimental Study in Beagle Dogs.

OBJECTIVE: To investigate the changes of the bacterial community in the oral environment of beagle dogs to gain insights on the possible causes of failed therapy in peri-implantitis. METHODS: Beagles were used as models for experimental peri-implantitis. Samples from peri-implant soft tissue (supramargin and submargin), ligature and contaminated surface of peri-implantitis sites were collected and analysed by sequencing the bacterial 16S rRNA gene. RESULTS: The residual microbial community from the curettes-treated implant surface contained a variety of microorganisms, including periodontal pathogens, which showed no changes in their composition and structure. CONCLUSION: It is possible that the residual bacterial community remained unchanged and this was the cause of recurrent episodes of inflammation.

Longitudinal Quantitation of Tooth Displacement in Chinese Adolescents with Normal Occlusion.

This longitudinal study aims to analyze the different modes of the maxillary and mandibular tooth displacement in subjects, who were aged 12.5-17.5 years (150-210 months), with untreated normal (Class I) occlusion. Longitudinal lateral cephalograms for a set of 10 subjects (7 females and 3 males) at consecutive annual time points were selected and monitored. Data were analyzed on the basis of the superimpositions of serial tracings of lateral cephalograms on stable anterior cranial base, the anatomies of the maxillary and mandibular structures. The horizontal and vertical displacements of the first molar and incisor were assessed by t-test. The local and the secondary tooth displacements with growth contributed to the total horizontal and vertical displacements of the molars and incisors of the subjects. In the total tooth displacement, the horizontal growth of maxilla and mandible had the same contribution as the local tooth displacements. The vertical maxillary growth played a smaller role than the local drift, and mandibular remodeling went in a reverse direction with the local tooth drift. The first molars moved more forward than the incisors in the upper and lower arches. Both the upper and lower first molars showed forward tipping. The analysis of tooth displacement may be utilized in making orthodontic treatment plan, including anchorage or torque control.

Novel Mutation of Cleidocranial Dysplasia-related Frameshift Runt-related Transcription Factor 2 in a Sporadic Chinese Case.

BACKGROUND: Cleidocranial dysplasia (CCD) is an autosomal dominant disease that affects the skeletal system. Common symptoms of CCD include hypoplasia or aplasia of the clavicles, delayed or even absent closure of the fontanels, midface hypoplasia, short stature, and delayed eruption of permanent and supernumerary teeth. Previous studies reported a connection between CCD and the haploinsufficiency of runt-related transcription factor 2 (RUNX2). Here, we report a sporadic Chinese case presenting typical symptoms of CCD. METHODS: We made genetic testing on this sporadic Chinese case and identified a novel RUNX2 frameshift mutation: c.1111dupT. In situ immunofluorescence microscopy and osteocalcin promoter luciferase assay were performed to compare the functions of the RUNX2 mutation with those of wild-type RUNX2. RESULTS: RUNX2 mutation was observed in the perinuclear region, cytoplasm, and nuclei. In contrast, wild-type RUNX2 was confined in the nuclei, which indicated that the subcellular compartmentalization of RUNX2 mutation was partially perturbed. The transactivation function on osteocalcin promoter of the RUNX2 mutation was obviously abrogated. CONCLUSIONS: We identified a sporadic CCD patient carrying a novel insertion/frameshift mutation of RUNX2. This finding expanded our understanding of CCD-related phenotypes.

Osteoprotegerin Promotes Cementoblastic Activity of Murine Cementoblast Cell Line in vitro.

OBJECTIVE: To investigate the effect of osteoprotegerin (OPG) on the cementoblastic activity of a clonal population of immortalised murine cementoblasts (OCCM-30) in vitro. METHODS: OCCM-30 cells were transiently transfected with the mouse OPG using the Avalanche transfection reagent. The ectopic expression of OPG was confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. The cell counting Kit-8 assay was used to investigate the effect of OPG on cell proliferation. The expression levels of cementoblastic-related mRNA and protein in the transfected OCCM-30 cells were detected using real-time PCR, Western blotting and immunohistochemical staining. RESULTS: Satisfactory transfection efficiency was observed 48 h after transfection. The results of the cell proliferation assay indicated that the expansion rate of the OPG transfection group was greater than that of the control group at both 72 h and 96 h. The mRNA levels of osterix (Osx), protein kinase B (Akt1), cementum attachment protein (CAP) and osteopontin (Opn) were significantly upregulated (P < 0.05) in the OPG group. Protein levels of OPN, bone sialoprotein II (BSP II), osteocalcin (OC) and CAP, which are responsible for osteogenetic and cementoblastic activity, were significantly increased in the OPG-overexpressing group. CONCLUSION: Overexpression of OPG in OCCM-30 cells promotes cementoblastic activity.

[A novel TaqMan® MGB probe for specifically detecting Streptococcus mutans].

OBJECTIVE: To design a new TaqMan® MGB probe for improving the specificity of Streptococcus mutans's detection. METHODS: We extracted six DNA samples from different streptococcal strains for PCR reaction. Conventional nested PCR and TaqMan® MGB real-time PCR were applied independently. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. The TaqMan® MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nested PCR. Streptococcus mutans DNA with 2.5 mg/L was sequentially diluted at 5-fold intervals to 0.16 µg/L. Standard DNA samples were used to generate standard curves by TaqMan® MGB real-time PCR. RESULTS: In the nested PCR, the primers specific for Streptococcus mutans also detected Streptococcus gordonii with visible band of 282 bp, giving false-positive results. In the TaqMan® MGB real-time PCR reaction, only Streptococcus mutans was detected. The detection limitation of TaqMan® MGB real-time PCR for Streptococcus mutans 16S rRNA gene was 20 µg/L. CONCLUSION: We designed a new TaqMan® MGB probe, and successfully set up a PCR based method for detecting oral Streptococcus mutans. TaqMan® MGB real-time PCR is a both specific and sensitive bacterial detection method.

Comparison of Chinese and white Bolton standards at age 13.

OBJECTIVES: To create a combined male-female Chinese Bolton standard for age 13 and to compare it to the combined Bolton standard for white 13 year olds. MATERIALS AND METHODS: Thirty-two Chinese and 32 white subjects (16 adolescent boys and 16 adolescent girls aged 13 years in each ethnic group) who fulfilled the selection criteria used to create the original Bolton standards were enrolled. Their cephalograms were traced and then averaged two at a time following the Bolton method. In addition to creating the template from the 32 subjects in each group, 43 linear and angular variables were measured and compared between the two ethnic groups and between the two sexes in each ethnic group using independent t-tests. A P value of .05 was used to assign statistical significance. The male-female combined templates for 13 year olds were also compared superimposing either in the Bolton relation or by soft tissue. RESULTS: No sexual dimorphism was found in the white subjects, whereas the Chinese girls had decreased upper face height (Na-ANS), more protrusive incisors (SNA-U1), and shorter cranial base (Ba-Na) and posterior cranial base (S-Ba) compared with the Chinese boys. In terms of ethnic comparison, the Chinese subjects presented shorter sagittal facial dimensions, a clockwise rotated mandible with a more acute gonial angle, and a convex facial profile with a less prominent nose and chin. However, overall vertical dimensions were similar. CONCLUSIONS: A 13-year-old combined male-female Chinese Bolton standard was created that demonstrated visually and with metric comparisons that Chinese and white clinically normal subjects have different craniofacial characteristics. These differences should be taken into consideration when an individualized orthodontic treatment plan is developed.

[Indication of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in gingival crevicular fluid to remodeling of alveolar bone during retention].

OBJECTIVE: To investigate the level of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in gingival crevicular fluid (GCF) during orthodontic retention and corresponding expression of OPG and RANKL in periodontal tissues. METHODS: Fifteen male Wistar rats (age, 6 weeks) were divided into three groups with 5 rats for each. GCF samples were collected at the baseline, 14 days after orthodontic force application, and 14 days after orthodontic force removal. enzyme linked immunosorbent assay (ELISA) was used to quantify the concentration of OPG and sRANKL (soluble receptor activator of nuclear factor kappa B ligand) in GCF. Immunohistochemical staining was performed to identify the OPG and RANKL expression in periodontal tissues. RESULTS: The concentration of sRANKL in GCF increased statistically significant from baseline to T2 (P<0.05) while decreased significantly from T2 to T3 (P<0.05). The concentration of OPG had minimum fluctuation from baseline to T3 without any statistical significance (P>0.05). The sRANKL/OPG ratio in GCF and that in periodontal tissues during the three time points were similar which showed a prominent increase in T2 (P<0.05 and P<0.01, respectively) and sharp decrease in T3 (P<0.05). CONCLUSION: sRANKL/OPG ratio may be one of the predictors reflecting the remodeling of periodontal tissues in orthodontic retention.

Longitudinal changes in mandibular arch posterior space in adolescents with normal occlusion.

INTRODUCTION: The purpose of this study was to investigate the changes of available mandibular space in the posterior dental arch of teenagers from 13 to 18 years old. METHODS: Longitudinal cephalograms of 28 adolescents (13 boys, 15 girls) with normal occlusion, selected from among 901 candidates, were taken annually from 13 to 18 years of age inclusively. Modified analyses with occlusal plane and occlusal plane perpendicular as reference planes were used to evaluate the changes of available space of the posterior mandibular arch. RESULTS: From 13 to 18 years of age, significant differences of mandibular posterior space were found among ages and sexes. The total increases of available space were 5.12 mm in the girls and 5.79 mm in the boys. For girls before age 16 and boys before age 17, the increased available space was contributed mainly by resorption of bone on the anterior border of the ramus. Mesial drift of the dental arch did not occur until the eruption of the third molars. The average available spaces increased 1.22 mm in girls less than age 16 and 1.45 mm in boys less than age 17 per side per year. CONCLUSIONS: The prediction of available space in the posterior mandibular arch should be based on age and sex.

[Intruding upper incisors using mini-screw anchorage in patients with gummy smile].

OBJECTIVE: To evaluate the effectiveness of mini-screw anchorage on intruding upper incisors in patients with gummy smile. METHODS: Sixteen female patients (mean age: 23.8 years) were involved in this study. The mini-screws were placed between upper lateral incisors and canines on both sides for intruding upper incisors together with fixed appliance. The cephalometric analysis was carried out. RESULTS: Totally 63 teeth in 16 patients were intruded successfully and the deep overbite was decreased by (2.74 +/- 2.00) mm (P < 0.001). The distance from the edge of the upper incisor to palatal plane was reduced by (5.00 +/- 2.76) mm (P < 0.001), and the incisors were proclined by (9.34 +/- 10.06) degrees (P < 0.01). The duration for intrusion was 10.8 months on average. CONCLUSIONS: It was practicable and effective to improve gummy smile by intruding upper incisors with mini-screw implant.

[Development and preliminary application of orthodontic friction dynamic testing apparatus].

OBJECTIVE: To design and assemble an orthodontic friction prototype testing apparatus and evaluate its performance. METHODS: Simulating progressive tooth tipping movement, results of the operating friction trials are reported at different archwire-bracket angulation. RESULTS: An orthodontic friction testing apparatus was designed and developed, by which the complexity of tooth movements observed with in vivo sliding mechanics was simulated and a series of in vitro friction experiments were conducted. This friction testing apparatus enables previously unattainable testing of the orthodontic archwire-bracket-ligation interface. In the passive configuration or the active configuration with second-order angulation, the friction in sliding mechanics was measured. CONCLUSION: The apparatus presented has the ability to allow for a high standard of basic hypothesis testing, product development and performance evaluation with relative ease. Furthermore, it will be the prototype of a computer-controlled multifunctional friction testing device in the near future.

[Accuracy and sensibility of computer -aided alveolar bone density quantitative measuring system].

OBJECTIVE: The alveolar bone density quantitative measuring system was established on the basis of grey level measuring on standardized-exposed direct digital periapical radiograph by referring to an aluminum step wedge with the aid of computer technique. We tested it's accuracy and sensibility by measuring a series of specimens and made it possible to estimate the area density of hydroxyl-phosphorite (HP) in the alveolar bone with the system. METHODS: The average grey level intensity of 19 specimens containing different amount of HP within certain area was measured with the system. The equivalent aluminum thickness (EAT) and corrected grey level intensity (CGL) of each specimen were calculated according to known grey level intensity of the aluminum step wedge and it's corresponding thickness. The liner regression equations between EAT, CGL and HP density (mg/mm2) were established. We put the values of EAT and CGL into the equations and calculated the corresponding values of estimated HP density. The bias ratios of estimation were then calculated. We measured two randomly chosen specimens 10 times respectively to calculate the system's measurement error range, then the sensibility of the system for measuring HP density was calculated according to the regression equations. RESULTS: EAT and CGL were significantly correlated with HP density. When EAT was larger than 0.67 mm or CGL larger than 41, the system's bias ratio of estimated HP density was lower than 5%. When we estimated the HP density with the use of EAT, the system could effectively check out a HP density change larger than 0.17 mg/mm2, with the use of CGL, a HP density change larger than 0.18 mg/mm2 could be effectively checked out. CONCLUSION: The accuracy and sensibility of the quantitative alveolar bone density measuring system are acceptable, it can be widely used to compare alveolar bone density longitudinally and cross-sectionally.

[Craniofacial characters for Angle's class II malocclusion using Procrusts superimposition and multivariate statistics].

OBJECTIVE: Cluster and discriminant analysis of the morphological characters of Angle's Class II malocclusion was performed using Procrusts standardization. The classification of craniofacial morphology with the help of morphometry and multivariate statistics was also discussed. METHODS: A total of 894 class II patients were collected from the department of orthodontic, Peking University School and Hospital of Stomatology during 1997-2000. Using Procrusts standardization and cluster analysis, the samples were divided into different groups, discrimination equations were then established. RESULTS: The samples were divided into 11 subdivisions by Procrusts superimposition and cluster analysis. Three discrimination equations were established. The accuracy rate of cross-validated grouped cases was 80.17%. CONCLUSION: Procrusts standardization had certain advantages in morphological classification; cluster analysis could be used in classification of Angle class II malocclusion; For different types, the differentiate rate was not the same; the discrimination equations was the foundation for future research.

[Bone morphogenetic protein-4 regulates embryonic tongue morphogenesis].

OBJECTIVE: To analyze the effect of Bone morphogenesis 4 and its antagonist Noggin on morphogenesis of tongue. METHODS: Dissected rats to get embryonic day 13 (E13) tongues; fed E13 tongues in standard medium, BMP4 (0.03 mg/L, 0.3 mg/L, 1 mg/L), and the antgonist Noggin(1 mg/L, 3 mg/L, 10 mg/L) medium; cultured for 3 days; fixed samples, observed tongues with scanning electronic microscope (SEM); measured the whole tongue length, anterior 1/8, 1/4 width and middle width of cultured tongues and analyzed data with SPSS 10.0. To further study the effects of BMP4 on epithelial and mesenchymal cell proliferation, Affi-gel blue gel beads were applied. Beads were soaked in PBS and BMP4 (667 mg/L), and implanted in the E13 embryonic tongues; then after cultured in standard medium for 3 days, tongues were embedded in O.C.T. and cut into 12 microm series sections. Ki67 was detected by immunohistochemical method. RESULTS: (1)Whole length of tongues changed greatly (P<0.05), the length was shortened in BMP4 groups (0.03 mg/L group 877.3+/-67.6 microm, 0.3 mg/L group 838.5+/-88.9 microm, 1 mg/L group 718.7+/-38.6 microm) compared with standard medium (1 037.8+/-126.2 microm), Noggin groups had no obvious change; the anterior 1/8 width of tongues changed significantly(P<0.05), the anterior 1/8 width was narrower in BMP4 groups (0.03 mg/L group 332.1+/-80.9 microm, 0.3 mg/L group 305.1+/-51.3 microm, 1 mg/L group 276.9+/-45.9 microm) compared with standard group(639.1+/-106.2 microm), except 10 mg/L group, Noggin groups were wider (1 mg/L group 815.5+/-90.3 microm, 3 mg/L group 857.6+/-87.1 microm, 10 mg/L group 807.1+/-113.8 microm); the anterior 1/4 width of tongue changed magnificently, also(P<0.05), BMP4 groups were narrower (0.03 mg/L group 421.3+/-43.8 microm, 0.3 mg/L group 407.3+/-15.6 microm, 1 mg/L group 363.7+/-24.7 microm) compared with standard group (653.7+/-101.6 microm), whereas, Noggin groups were wider greatly (1 mg/L group 838.0+/-130.5 microm, 3 mg/L group 947.2+/-34.9 microm, 10 mg/L group 889.4+/-74.6 microm); the middle width of tongue changed significantly(P<0.05), width of BMP4 groups were narrower (0.03 mg/L group 567.3+/-35.8 microm, 0.3 mg/L group 548.4+/-30.5 microm, 1 mg/L group 457.4+/-48.0 microm) compared with standard medium (683.1+/-79.8 microm), and Noggin groups had widening tendency, difference in 3 mg/L group is magnificent (1 mg/L group 776.2+/-134.1 microm, 3 mg/L group 964.3+/-44.3 microm, 10 mg/L group 777.2+/-46.7 microm). (2) The expression of Ki67 in both epithelium and mesenchym adjacent to BMP4 beads reduced greatly. CONCLUSION: BMP4 could effect morphological development of embryonic tongue, which could change spatula-shape tongue into short, narrow and tip-point one, the antagonist Noggin tongue was wider and longer; BMP4 inhibit cell proliferation in embryonic tongues.

[Preliminary investigation of Tip-Edge Plus technique in the treatment of Class III malocclusion].

OBJECTIVE: To investigate the craniofacial and occlusal changes of Class III cases treated with Tip-Edge Plus Technique. METHODS: Thirteen Class III patients (6 girls and 7 boys, mean age 14.9 years) with mandibular protrusion were selected. All the patients were four premolar extraction cases and treated with Tip-Edge Plus technique. Cephalometric analysis was performed to evaluate the changes before and after treatment. RESULTS: After treatment, Wits changed from (-2.50 +/- 1.40) mm to (-1.00 +/- 0.85) mm and ANB changed from (-0.61 +/- 1.41) degrees to (1.31 +/- 1.66) degrees (P < 0.05). UL-E and LL-E were decreased significantly from (7.55 +/- 1.24) mm to (4.82 +/- 1.05) mm and from (10.16 +/- 2.43) mm to (6.03 +/- 2.01) mm respectively (P < 0.01). The incisors were retracted. A remarkable soft tissue change was noted after the treatment. The difference between the distance of the upper lip and lower lip to SnPg' at the beginning of treatment changed from (-1.57 +/- 1.24) mm to (0.62 +/- 1.24) mm (P < 0.01). FCA changed from (2.62 +/- 1.53) degrees to (7.02 +/- 2.21) degrees . CONCLUSIONS: With Tip-Edge Plus straight-wire technique and light continuous force, anterior teeth could be tipped distally and lingually quickly. The hard and soft tissues changed accordingly.

[Analysis of treatment templates of Angle's Class III malocclusion patients].

OBJECTIVE: To analyze the morphological characters of Angle's Class III malocclusion patients and the relationship between characters and treatment options. METHODS: A total of 472 Class III patients were collected during 1997-2000. The standardized coordinates' values of the patients were obtained by Procrustes superimposition (PS). Cluster analysis was carried out to divide the patients into different subdivisions and the relationship between treatment options and subdivisions was analyzed. RESULTS: 472 patients were divided into 14 subdivisions with special characters. The morphological templates were formed by computer. There were certain relationships between characters and treatment options. CONCLUSION: The mechanism of malocclusion of different Class III patients are not necessarily same. The typing has significance to choose the treatment method.

Quantitative cervical vertebral maturation assessment in adolescents with normal occlusion: a mixed longitudinal study.

INTRODUCTION: The purpose of this study was to establish a quantitative cervical vertebral maturation (CVM) system for adolescents with normal occlusion. METHODS: Mixed longitudinal data were used. The subjects included 87 children and adolescents from 8 to 18 years old with normal occlusion (32 boys, 55 girls) selected from 901 candidates. Sequential lateral cephalograms and hand-wrist films were taken once a year for 6 years. The lateral cephalograms of all subjects were divided into 11 maturation groups according to the Fishman skeletal maturity indicators. The morphologic characteristics of the second, third, and fourth cervical vertebrae at 11 developmental stages were measured and analyzed. RESULTS: Three characteristic parameters (H4/W4, AH3/PH3, @2) were selected to determine the classification of CVM. With 3 morphologic variables, the quantitative CVM system including 4 maturational stages was established. An equation that can accurately estimate the maturation of the cervical vertebrae was established: CVM stage=-4.13+3.57xH4/W4+4.07xAH3/PH3+0.03x@2. CONCLUSIONS: The quantitative CVM method is an efficient, objective, and relatively simple approach to assess the level of skeletal maturation during adolescence.

[The differences of hard tissue changes and stability of Angle's Class II division 1 extraction cases treated by Begg appliance and Edgewise appliance].

OBJECTIVE: The purpose of this investigation is to compare the differences of hard tissue changes and stability in patients with Class II division 1 treated with extraction of four first premolars by Begg appliance and Edgewise appliance. METHODS: The study was conducted using lateral cephalometric radiographs taken pretreatment, posttreatment and following-up. Thirty patients who had an Angle's Class II division 1 malocclusion were treated with first premolar extractions (19 by Begg appliance and 11 by Edgewise appliance). Cephalometric radiographs were scanned and the data were analyzed according to the paired and unpaired t-test. RESULTS: The differences between the pretreatment and posttreatment periods were statistically significant for all the incisor and molar cephalometric variables (P<0.05) except UMA-PPV and UMC-PPV by Edgewise appliance. During treatment the incisors moved lingual-inclined and extrusively, the molar moved mesially and extrusively except that the upper molar of the Edgewise group stayed relatively stable anteroposteriorly. During the follow-up period the anterior teeth moved forward and extrusively, the upper molar moved mesially and extrusively. There was no significant differences of tooth movement and stability between Begg appliance and Edgewise appliance (P>0.05). CONCLUSION: There is no significant differences of hard tissue changes and stability between Begg appliance and Edgewise appliance.