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Objective: To investigate the effects of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on collagen phagocytosis of human periodontal ligament fibroblasts (hPDLF)., Methods: Fluorescence localization and flow cytometry was used to test the collagen phygocytosis of hPDLF stimulated by the various concentration of LPS for 48 hours in vitro., Results: The collagen phygocytosis of hPDLF was increased significantly by 0.1 microg/mL LPS (P < 0.05)., Conclusion: This study indicates that P. gingivalis LPS may enhance the degradation of collagen by stimulating the phagocytic activity of the hPDLF in periodontitis.

Objective: To determine whether the glucan binding protein C (GbpC) with LPXAG motif is anchoring to the cell wall of the Streptococcus mutans UA159 (S. mutans UA159)., Methods: S. mutans UA159 GbpC C terminal gene segment was amplified by PCR, the gene sequences and the deduced amino acid sequences were analyzed. In order to locate the GbpC of S. mutans, the study isolated the wall fraction following digestion of the cell wall by N-acetylmuramidase, and the GbpC was detected by Western blot analysis. GbpC S. mutans UA159 was located with gold particles. Furthermore, the dextran-dependent aggregation (ddag) phenotype of the S. mutans UA159 under stress condition was observed., Results: S. mutans UA159 GbpC C-terminal LPXTG motif was replaced by LPXAG motif. GbpC was observed in the cell wall component and immunogold experiment showed that the gold particles distributed around the cell wall surface. S. mutans UA159 exhibited ddag+., Conclusions: GbpC with LPXAG motif was still anchoring to the cell wall.

Objective: To investigate the effect of retinoic acid on differentiation of periodontal ligament cells (PDLCs) and gingival fibroblasts (GFs)., Methods: The periodontal ligament cells and gingival fibroblasts were cultured, challenged with different concentrations of retinoic acid in medium and detected for the alkaline phosphatase (ALP) activity and its mRNA by biochemical technique, in situ hybridization and RT-PCR., Results: ALP activity in normal PDLCs was higher than that in GFs. The mRNA signals were positive in PDLCs, and negative in GFs. After treated with different concentrations of retinoic acid, ALP activity of PDLCs was increased than that of the control, and its mRNA signals were enhanced, especially in 5 x 10(-6) mol/L. The treated GFs showed a slight increase of ALP activity and a weak band of mRNA signals only in 5 x 10(-6) molUL concentration., Conclusion: There were differences between PDLCs and GFs in differentiating into osteoblast-like cells.

Objective: To investigate root canal curvature in maxillary anterior teeth., Methods: About 400 human maxillary anterior teeth were examined by indirect digital radiography both from labiolingual and mesiodistal directions. The root canal curvature was analyzed., Results: The major sort of maxillary anterior teeth was type I. The proportion of maxillary center incisors, maxillary lateral incisors, maxillary canines curved in mesiodistal were 11.17%, 16.19% and 21.43%, in labio-lingual were 39.59%, 40.00% and 29.59%, both in labiolingual and mesiodistal directions were 4.60%, 35.24% and 24.49%. About 50% of maxillary anterior teeth were moderate curve, the degree of root canal curvature of maxillary canines was larger than that of maxillary incisors, and radius of curvature and length of the curved part of root canal of maxillary canines were smaller than that of maxillary incisors., Conclusion: Root canal curvature in maxillary anterior is complex, especially in maxillary canine. In order to improve quality of root canal therapy, we need to understand types of root canal, degree of root canal curvature and radius of curvature completely.

Objective: To assess the difference of genetic alteration patterns among different areas in the same oral squamous cell carcinoma (OSCC)., Methods: Studied the loss of heterozygosity (LOH) and microsatellite instability (MI) at chromosomal loci TP53 and RPS6 on the invasive tumor front (ITF), the center/superficial part and stroma cells by combining laser capture microdissection (LCM) and PCR technique., Results: There existed a high frequency of LOH and MI on chromosomes loci TP53 and RPS6. The frequency of RPS6 and TP53 aberration at the stroma was 23.5% (4/17) and 43.8% (7/16), respectively. While in epithelial part (both ITF and center), it reached up to 64.7% (11/17) and 70.6% (12/17) respectively, and the difference was significant (P < 0.05). The overall frequency of the two markers was statistically higher at the ITF (20/32) than at the center/superficial part (15/34) (P < 0.05)., Conclusions: The current study revealed that genetic alterations were different in different areas of the same tumor and there existed a relationship between the histological grading and genotypes of OSCC.

Purpose: To compare the cleaning efficacy and shaping ability of engine-driven ProTaper and GT files, and manual preparation using K-Flexofile instruments in curved root canals of extracted human teeth., Methods: 45 canals of maxillary and mandibular molars with curvatures between 25 degrees and 40 degrees were divided into three groups. The groups were balanced with regard to the angle and the radius of canal curvature. Canals in each group were prepared to an apical size of 25 with either the rotary ProTaper or GT system, or manually with K-Flexofile using the modified double-flared technique. Irrigation was done with 2 mL 2.5% NaOCl after each instrument and, as the final rinse, 10 mL 2.5% NaOCl then 10 mL 17% EDTA and finally 5 mL distilled water. The double-exposure radiographic technique was used to examine for the presence of apical transportation. The time required to complete the preparation, as well as any change in working length after preparation were recorded. The roots were then grooved and split longitudinally. The amounts of debris and smear layer were evaluated at the apical, middle and coronal regions under the scanning electron microscope. Data were analyzed either parametrically with the F-test or non-parametrically using the Kruskal-Wallis test, where appropriate., Results: Two GT files but none of the K-Flexofile and ProTaper instruments separated. For debris removal, the ProTaper group achieved a better result than GT (P < 0.05) but not the K-Flexofile group at all three regions (apical, middle and coronal). K-Flexofiles produced significantly less smear layer than ProTaper and GT files only in the middle third of the canal (P < 0.01). Both NiTi rotary instruments maintained the original canal shape better than the K-Flexofiles (P < 0.05) and required significantly less time to complete the preparation.

Objective: To compare the initial penetration depth of nickel-titanium (NiTi) and stainless-steel (SS) spreader during lateral compaction and the quality of the seal in curved canals., Methods: Forty extracted mandibular premolars with a single curved canal were divided into two groups: no more than 20 degrees and more than 20 degrees based on degree of curvature. All canals were instrumented using a rotary instrumentation technique. NiTi and SS spreaders were used to obturate the canals containing a master cone while the penetration depths were measured. Horizontal sections were cut in 2 and 4 mm from the apex and photographed under stereomicroscope. The percentage of gutta-percha-filled are (PGP) of cross-sections was measured using an image analysis program., Results: In canals of more than 20 degrees, the penetration depths and PGP of 2 mm from the apex of NiTi spreaders were higher than SS spreader. In canals of no more than 20 degrees, there were no significant difference between them (P > 0.05). At 4 mm from the apex, there was no significant difference between two groups., Conclusion: NiTi spreaders has a higher penetrated depth and obturation density than SS spreaders in severed curved canals.

Objective: To assess the clinical outcome of root canal therapy (RCT) and the various factors that may influence the outcome of RCT., Methods: A total of 695 teeth from 357 patients were retrospectively studied three years after endodontic treatment. Pre- and intra-operative information was collected from the original patient records. The post-operative sign or symptom, periapical status and coronal restoration integrity were examined 3 years after obturation. Data were subjected to bivariate and multivariate analysis., Results: The cure rate for 695 teeth was 75.1%, 96.0% of which was considered to be functional. The tooth group, pre-operative pulp and periapical status, quality of root filling and integrity of coronal restoration were revealed by means of bivariate analysis to exert a significant influence on treatment outcome. The logistic analysis indicated that the odds for cure in the teeth with pre-operative periapical radiolucency, underfilling and "open" coronal restoration were significantly lower by 2 folds, 3 folds and 1.6 folds than their counterparts, respectively., Conclusions: The pre-operative periapical status, quality of root filling and the integrity of coronal restoration are main predictors of outcome in RCT.

Purpose: The purpose of this study was to compare the salivary immunoglobulin A antibody response to Streptococcus mutans in normal with in heat treated stress., Methods: Clinical Streptococcus mutans strains were isolated from 20 volunteers, serotyped by biochemical test and PCR, and genotyped by AP-PCR. Unstimulated secretions from submandibular glands and sublingual glands were collected from volunteers by modified collectors. Each identified genotype was cultured in two groups: control group was grown in BHI broth at 37 degrees C. for 3 hours; stress group was incubated in BHI broth at 42 degrees C. for 3 hours. Analysis of SIgA activity to clinical genotype strains and reference strains in different group was detected by Western blot., Results: There was no significant difference between stress group and control group,in spite that some bands had strong or weak intensity. Different genotypes of S.mutans could have different immunoblotting profile as for an individual. SIgA from different volunteers could have different immonoblotting profiles as to the same genotype strain., Conclusions: Although Streptococcus mutans can express heat shock proteins in stress, this study suggests these new proteins have no significant effect on the reaction of SIgA to Streptococcus mutans. Different genotype strains may have different proteins, and different immunoreactivity to host. Different hosts may have different immunoreactivities to one genotypes of S.mutans.

Objective: To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells., Methods: The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants., Results: Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX., Conclusions: CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

Objective: To construct fluorescent eukaryotic cell expression vector with human bone morphogenetic protein-7 (hBMP-7) gene and to transfect mouse stromal cell line W-20-17 to detect the bioactivity of pEGFP-hBMP-7 in vitro., Methods: pEGFP-hBMP-7 plasmid was constructed by subcloning technique and identified by enzyme cutting and electrophoresis. W-20-17 cells were transfected with pEGFP-hBMP-7 by means of lipofectamine-2000 media methods. Transfection efficiency and gene expression were evaluated by fluorescent microscopy. ALP, von Kossa and osteocalcin (OC) were tested to determined the phenotypes of osteoblast., Results: After 48 hours, the gene transfection efficiency was 40%. Based on GFP and immunofluorescence of pEGFP-hBMP-7, there was the expression of aim gene. After gene transfection, there were not significant different of cell morphology feature and cell proliferation. ALP activity, the number of calcium nodules and the expression of OC increased., Conclusions: pEGFP-hBMP-7 with bioactivity was constructed, which could induce W-20-17 cells to differentiate to osteoblasts.

Objective: To determine the physicochemical properties of the mutanase of Trichoderma harzianum isolated from China and to study the influence of mutanase on the adherence of oral Streptococci and the structure of oral biofilms., Methods: Six fungal strains belonging to Trichoderma were tested for mutanase production in the same cultural condition, the strain producing the highest mutanase activity was studied further and the pH and temperature optimum of the enzyme was determined. The RT-PCR method was used to obtain the gene coding for mutanase and the product was cloned to pMD18-T simple vector for sequencing. Inhibition effects of mutanase on the adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 were studied by adherence test. The optical sectioning of biofilms with or without mutanase supplementation were analyzed by confocal laser scanning microscopy (CLSM)., Results: The highest enzymatic activity was achieved by Trichoderma harzianum Th1, the maximum activity was at pH 5.5 and at 40 degrees C. The nucleotide sequence was 92% homology with that of a known gene coding a mutanase (GenBank accession No. AJ243799). The adherence of Streptococcus sobrinus OMZ176, Streptococcus sobrinus 6715, Streptococcus mutans MT8148 was significantly inhibited by mutanase. Compared with control, the biofilms with mutanase supplementation had lower height and sparser structure., Conclusions: The mutanase from Trichoderma harzianum Th1 can inhibit the adherence of oral Streptococci and had an influence on the structure of oral biofilms.

Objective: To investigate the expression of transcription regulator LMO4 mRNA in the developing mouse molar and compare the expression pattern of LMO4 with that of Shh signaling molecule., Methods: Wild-type embryos used in this study (E11.5-P1.5) were generated by mating Kun-Ming mice. The expression pattern of LMO4 during organ development was carried on by whole-mount in situ hybridization. The expression patterns of LMO4 and Shh mRNA during molar development were analysed by section in situ hybridization. Immunohistochemical staining of PCNA was carried on by SP method., Results: LMO4 mRNA was widespread at early embryonic stages (E11.5) with positive hybridization signal in the mandibular reason, limb bud, brain, epidermis and somites revealed by whole-mount in situ hybridization. Section in situ hybridization showed that LMO4 was expressed in the tooth bud, the two tips of the enamel organ and the cervical loop from E13.5 to E16.5. While Shh was localized in the enamel knot on E14.5. On E18.5-P1.5, LMO4 transcripts were distributed in the ameloblast and the stratum intermedium. On E13.5-E16.5, the tooth bud cells and the cervical loop cells were PCNA positive. These were the same regions that showed LMO4 mRNA expression., Conclusions: LMO4 was confined to the dental epithelium and had spatial temporal expression patterns during tooth morphogenesis. The expression patterns of LMO4 and Shh were similar. In early tooth development, LMO4 might regulate cell proliferation. In late tooth development, it might participate in the ameloblast differentiation.

Objective: This research aimed to study and assess clinical application of the using of three Nickel-titanium (NiTi) rotary instruments, namely ProFile, ProTaper and Hero 642, in preparation of curved root canals., Methods: 80 teeth with curved root canals were instrumented by ProFile, ProTaper or Hero 642 rotary instruments using crown-down technique in test groups and by stainless steel K files in control group. All teeth were obturated with lateral condensation method. The efficiency of preparation and obturation was analyzed with radiographs before, during and after operation., Results: Three NiTi rotary instruments could keep the curvature and flow of curved canals very well, and the continuously tapered root canal shape was achieved. There were no transportation, apical blockage, and ledge in all test groups. The operative time was shorter for ProTaper than that of ProFile and Hero 642. Endodontic flare-up seldom occurred in test groups. Two rotary instruments fractured in canals., Conclusion: With the NiTi rotary instruments, ProFile, ProTaper and Hero 642, curved root canals can be prepared effectively and quickly. Meanwhile, the problem of instrument separation should be prevented.

Objective: To describe clinical and histological features of oral granular cell tumor (OGCT)and discuss their proliferative activity., Methods: Clinical and microscopic features were assessed in 14 cases of OGCT collected from the department of oral pathology, college of stomatology of Wuhan University between 1970 and 2003. Immunohistochemical analysis was carried out using antibodies to S-100, NSE and Ki-67 and follow-up was obtained in all cases., Results: Tongue was the most commonly affected location (13/14). The average age was 32.6 years (range 11 to 50). OGCT occurred more commonly in females (2.5:1). Histologically, the lesions consisted of polygonal cells with abundant, granular cytoplasm. Eleven cases had typical histological features, while 3 specimens were atypical. Growth patterns were expansive in 3/14 and invasive in 11/14, including 3 atypical cases. Immunohistochemical analysis disclosed that 100% of granular cells demonstrated moderated/strong staining for S-100 protein, neuron specific enolase (NSE). Nuclear immunostaining for Ki-67 was observed only in isolated granular cells. Seven patients with benign and two patients with atypical granular cell tumor had no recurrence and metastases. One patient with atypical granular cell tumor had local recurrence after 9 years and died of the disease 10 months later., Conclusions: OGCT cells display low proliferation activity. Most OGCTs are benign but few have malignant potential and periodic follow-up is mandatory to detect malignant transformation.

Objective: To isolate and cultivate human dental pulp stem cells (DPSCs)., Methods: Pulp tissue was removed from healthy young human teeth extracted for orthodontic purposes. The pulp was digested by Type I collagenase and dispase. Then single-cell suspensions were obtained by filter and cultivated. The clones were identified by expression of STRO-1. Under the conditions of inducement, clones were identified by activity of alkaline phosphatase (ALP), formation of mineralized nodule and expression of dentin sialoprotein (DSP), and by Oil Red-O dyeing and expressing of PPARr2., Results: The clones had positive expression of STRO-1. When stimulated to differentiation, these cells took on dramatically high activity of ALP, had the ability of mineralization and expressed DSP. These cells also had ability to trans-differentiate into adipocytes., Conclusion: There are stem cells in human dental pulp tissues, which can be isolated and cultivated.

Objective: To test the salivary immunoglobulin A antibody activity to Streptococcus mutans in normal with in acid environment., Methods: Streptococcus mutans strains were isolated from 20 volunteers, serotyped by biochemical test and PCR, and genotyped by AP-PCR. Unstimulated secretions from submandibular glands and sublingual glands were collected from volunteers by modified collectors. Each identified Streptococcus mutans genotype was cultured in two groups: control group was cultured in BHI broth pH7.2 at 37 degrees C for 2 h; acid shock group were cultured in TYEG broth (pH5.5) at 37 degrees C for 2 h. Analysis of SIgA activity to Streptococcus mutans genotypes in different groups was detected by Western blot., Results: (1) The SIgA of each individual could response to his own Streptococcus mutans strains and the reference strains; (2) The same individual had different SIgA activity to different genotype strains; (3) There were no significant difference between acid groups and control groups, in spite that some bands had strong or weak intensity., Conclusions: Although Streptococcus mutans could express acid shock proteins in stress, the present study suggests that these new proteins have no qualitative effect on the reaction of SIgA to Streptococcus mutans.

Objective: To investigate the effects of dexamethasone on Th1/Th2 cytokines in oral lichen planus., Methods: Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from OLP patients and healthy controls. PBMC from patients with OLP were stimulated with phytohemagglutinin (PHA) and dexamethasone respectively for 72 h. The concentrations of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in culture supernatants were determined by ELISA. The mRNA levels for IFN-gamma and IL-4 in culture cells were evaluated using semi-quantitative reverse transcription-polymerase chain reaction., Results: Compared with healthy controls, the levels of IFN-gamma in OLP patients were significantly lower (P < 0.05). The levels of IL-4 were higher, but not statistically significant (P > 0.05). The ratios of IFN-gamma/IL-4 were lower in patients with OLP (P < 0.05). Dexamethasone significantly inhibited the levels of IFN-gamma and IL-4 (P < 0.01). Moreover, IFN-gamma was inhibited significantly more than IL-4. The levels of IFN-gamma and IL-4 mRNA expression in culture cells were consistent with protein production in supernatants., Conclusions: Th2 immune response is predominant in OLP. Dexamethasone is an immunosuppressant inhibiting Th1/Th2 cytokines.

Objective: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in situ. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and pGLUA-P, a fusion anticaries DNA vaccine., Methods: pGJA-P was administrated intramuscularly or intranasally to rats, and the expression of recombinant protein was detected by immunohistochemistry technique. Wistar rats were fed a cariogenic diet and orally infected with S. mutans, then immunized with pGJA-P or pGLUA-P via the intramuscular or intranasal route. All rats received a booster immunization 2 weeks later. At the termination of the experiment, blood and saliva samples were collected for assay of antibodies by ELISA and jaws were obtained for caries evaluation by the Keyes method., Results: Recombinant protein could be detected in muscle in intramuscularly immunized rats and in nasal mucosa in intranasally immunized rats. Rats immunized intramuscularly with pGJA-P had significantly higher serum IgG levels than others (P < 0.01). Rats immunized intranasally or intramuscularly with pGJA-P had significantly higher salivary IgA levels than others (P < 0.01). Keyes scores of pGJA-P groups were significantly lower than those of pGLUA-P groups and pCI groups (P < 0.01)., Conclusions: pGJA-P could be correctly expressed in vivo. pGJA-P generated increased humoral immune response and anticaries efficacy compared with pGLUA-P.

Objective: To construct an acid-sensitive mutant of Streptococcus mutans (S. mutans) by transposon mutagenesis and to find a new gene related to the acid tolerance of S. mutans., Methods: The transposon Tn917 was delivered into S. mutans UA159 by the temperature-sensitive plasmid pTV1-OK bearing Tn917 and transposition of Tn917 was induced after incubation at non-permissive temperature (42 degrees C). Transposants harboring Tn917 in the chromosome were screened for the selection of mutant that had diminished growth at low pH. Southern analysis was performed with EcoRI (no cut within Tn917) digests of S. mutans UA159 and the selected aid-sensitive mutant, with DIG-labeled probe of 4.3 kb KpnI fragment of pTV1-OK containing Tn917. Genetic backcross experiment was performed by transforming the genome of the mutant to another S. mutans strain MT8148 to determine the linkage of Tn917 insertion to the change of phenotype (acid-sensitivity). Comparison of the abilities to grow at low pH, the glycolytic pH drop and killing pH values were done between the acid-sensitive mutant and the parent strain. The asymmetric PCR method was used to obtain the fragment flanking Tn917 and the PCR products were cloned to pMD18-T vector for sequencing., Results: One mutant that showed no growth at pH 5.0 was isolated from 2 316 transposants and was named as b23. Southern analysis and genetic backcross experiment confirmed the linkage between single Tn917 insertion into the chromosome and the phenotypic change (acid sensitive). b23 was less acid tolerant than UA159 in that it showed poorer growth at low pH and higher glycolytic pH minimum and higher killing pH. BLAST results indicated that Tn917 inserted into the genome of S. mutans UA159 at the site of 996 123 bp., Conclusion: An acid-sensitive mutant of S. mutans was successfully constructed and a new gene that is responsible for the acid tolerance in S. mutans UA159 was revealed.

Objective: To study the effect of the left bacteria on the root canal therapy., Methods: 50 single-rooted teeth with chronic apical periodontitis were divided into two groups, one was instrumented with step-back technique and 2.5%NaOCl ultrasonic irrigation for 3 min, then filled with Thermafil. Samples were taken after instrumentation to culture. The other was treated with traditional RCT at three visits., Results: In 24 months the apical radiolucency were greatly reduced in all cases. There weren't significant relationship among the postoperative pain and the left bacteria, the degree of the obturation or the pre-operative symptoms (P > 0.05)., Conclusion: The effect of left bacteria in root canal filled with Thermafil wasn't observed.

Objectives: To observe the expression of a targeted fusion anticaries DNA vaccine pGJA-P in muscular in vivo. To compare the levels of specific antibodies and anticaries efficacy generated by pGJA-P and fusion anticaries DNA vaccine pGLUA-P in gnotobiotic rats, and observe the kinetics of antibody responses in BALB/c mice., Methods: (1) Twelve 28-day-old female Wistar rats were randomly divided into 2 groups of 6 rats to be injected with the plasmid pGJA-P containing the signal peptide and extracellular regions of human CTLA(4), hinge and Fc regions of human IgG, the glu sequence of gtfB gene and A-P fragment of pac gene of Streptococcus mutans or the eukaryotic expression plasmid pCI into the quadriceps muscle of thigh respectively. Three days after the rats were killed and specimens of quadriceps muscles of thigh were taken. Immunohistochemical SP staining was used to examine the in situ expression of pGJA-P. (2) Twenty-four 18-day-old female Wistar rats were randomly divided into 4 groups of 6 rats. The rats were fed with cariogenic food. During the age of 20 - 22 days cariogentic food containing broad-spectrum antibiotics was fed. Then aseptic cotton stick was used to swab the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. During the age of 24 - 26 days, S. mutans Ingbritt cultured anaerobically was swab onto the surface of teeth of the rats twice with an interval of 30 minutes. After the inoculation aseptic cotton stick was used to wipe the oral cavity and be smeared onto the solid medium so as to observe the growth of bacteria under anaerobic culture for 48 hours. When the gnotobiotic rats were 28 days old they were injected with pGJA-P, pGLUA-P, fusion anticaries DNA vaccine against both PAc, cell surface protein antigen, and glucosyltransferase (GTF), pCI or normal saline into the quadriceps muscle of thigh respectively, 2 weeks later a booster shot was given. When the rats were 63 days old their saliva and blood samples were collected. The serum IgG and salivary IgA were assayed by using ELISA. The gnotobiotic rats were killed and their maxillary bone the mandibles were isolated. The anticaries effect was evaluated by Keyes caries scores. (3) Twenty-four 4-week-old BALB/c mice were randomly divided into 4 groups of 6 mice: to be injected with pGJA-P, pGLUA-P, pCI, or normal saline respectively into the quadriceps muscles of thigh, 2 weeks later a booster shot was given. Before the injection and every 2 weeks after the immunization specimens of saliva and blood were collected. The serum IgG and salivary IgA were assayed by using ELISA., Results: (1) Recombinant protein could be detected in the quadriceps muscles of the rats immunized with pGJA-P, but not in the muscles of the rats immunized with pCI. (2) The levels of serum anti-PAc IgG (1:200 000) and anti-GTF IgG (1:58 000) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:23 000 and 1:11 000 respectively) (both P < 0.01). The levels of salivary anti-PAc IgA (1:8) and anti-GTF IgA (1:6) of the rats immunized with pGJA-P were significantly higher than those of the rats immunized with pGLUA-P (1:2 and 1:2 respectively) (both P < 0.01). The Keyes scores of the pGJA-P group were significantly lower than those of the pGLUA-P group and the control groups (all P < 0.01). The effective serum IgG and salivary IgA in the pGJA-P group and effective serum IgG in the pGLUA-P group all persisted to the end of the experiment. (3) Two weeks after the initial immunization the serum anti-PAc IgG level of the mice immunized with pGJA-P increased remarkably, 4 times that of the mice immunized with pGLUA-P, and 33 times those of the mice injected with pCI or normal saline. Two weeks after the booster immunization, the serum anti-PAc IgG level of the mice immunized with pGJA-P was 14 times that of the mice immunized with pGLUA-P, and 117 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG immunized with pGJA-P reached its peak 10 weeks after the initial immunization, 4 times that of the mice immunized with pGLUA-P, and 160 times those of the mice injected with pCI or normal saline. The serum anti-PAc IgG of the mice immunized with pGLUA-P reached its peak at 16 weeks, however, significantly lower than the peak of the mice immunized with pGJA-P (P < 0.01). The serum anti-Pac IgG levels of the mice injected with pCI or with normal saline were not significantly different (P > 0.05). Since the second week after the initial immunization, significant difference in the serum anti-PAc IgG level could be seen between the mice immunized with pGJA-P or the mice immunized with pGLUA-P, and between the mice immunized with pGJA-P and the mice immunized with pGLUA-P and those injected with pCI or normal saline (all P < 0.01). Six weeks after the initial immunization the salivary anti-PAc IgA level of the mice immunized with pGJA-P was 18 times those of the mice injected with pCI or with normal saline (both P < 0.01), 10 weeks after the salivary anti-PAc IgA level of the mice immunized with pGJA-P reached its peak, 24 times those of the mice immunized with pCI or normal saline without a significant difference between the latter 2 groups (P > 0.05). No effective salivary IgA response was seen in the mice immunized with pGLUA-P., Conclusion: pGJA-P can be expressed in vivo. Immunization with pGJA-P intramuscularly induces effective mucosal and systematic humoral responses. It is an effective DNA vaccine against dental caries.

Objective: To detect the immunoreactivity of targeted fusion anti-caries DNA vaccine pGJA-P in vitro, and the ability to enhance the immune responses compared with the non-targeted fusion anti-caries DNA vaccine pGLUA-P., Methods: The CHO cells were transfected with pGJA-P and the expression of recombinant protein in cultured supernatants were detected using Western blotting. 5 to 6-month-old female Japanese rabbits were immunized with either pGJA-P or pGLUA-P via either intramuscular injection (i.m.) or intranasal route (i.n.). The sera and saliva were collected and the antibody responses were checked by ELISA. The effect of immune sera on the synthesis of water-insoluble glucan by glucosyltransferase of S. mutans was examined., Results: The expressed protein could response to specific anti-GTF antibody. The antibody responses in serum generated by pGJA-P via i.m. were significantly higher than those generated by pGLUA-P (P < 0.01). The antibody responses in saliva generated by pGJA-P via i.n. were significantly higher than those generated by pGLUA-P (P < 0.01). The higher mucosal antibody response induced by pGJA-P via i.m. compared with pGLUA-P (P < 0.01) was detected. The immune sera of rabbits immunized by pGJA-P via i.m. most significantly inhibited the synthesis of water-insoluble glucan by glucosyltransferase., Conclusions: The recombinant protein expressed by pGJA-P had the immunoreactivity to anti-GTF antibody. pGJA-P could induce faster and higher specific mucosal SIgA antibody responses via i.n. or serum IgG antibody responses via i.m. compared with non-targeted DNA vaccine, pGLUA-P. High titres of specific mucosal antibodies were found in rabbits immunized with pGJA-P via i.m. The immune sera of rabbits immunized by pGJA-P via i.m. displayed the ability of inhibiting the synthesis of water-insoluble glucan by glucosyltransferase.

Objective: To evaluate clinical effect of the new ProTaper Nickel-titanium rotary instruments in preparation of curved root canals., Methods: 68 teeth with curved root canals were instrumented by ProTaper rotary instruments using crown-down technique, and by K files using step-back technique for control. All teeth were obturated with lateral condensation method. The efficiency of preparation and obturation was analyzed with radiographs before, during and after operation., Results: No transportation, apical blockage and ledge were found in test. The technique could keep the curvature and flow of curved canals. There were two ledge and more apical transportation (P < 0.01) in control than in test. The operative time was shorter and post treatment pain seldom occurred in ProTaper group., Conclusion: The ProTaper NiTi rotary instruments can prepare curved root canals effectively and safely. It is an efficient instrumentation method for curved canals and can be used widely.

Objective: To analyze the clinical fracture of nickel-titanium instruments and its causes., Methods: 68 Profile and NiTiflex files broken during the clinical root canal instrumentations in two years were collected. The analysis was carried out regarding the relation between the broken files and the distribution in the teeth, canal, degree of curvature of canals, and the relation between amount of the broken files and the period., Results: The majority of the clinical fracture of nickel-titanium instruments were flexural fatigue which frequently occurred in molar, the midpoint of the curvature, canal with curves > 30 degrees and very complex canals. Amount of the broken nickel-titanium instruments in the first year was twice than that in second year., Conclusion: The fracture of nickel-titanium instruments is closely related to the anatomy of the root canal, the operator's experience and the usage of instrument and so on.

Objectives: To investigate the carious dissolution mechanism of enamel apatite nano-crystals at lattice fringe level and the possible correlation between the carious dissolution and the appearance of central dark line (CDL) in enamel crystals., Methods: The body of the lesion in incipient enamel caries was observed by high resolution transmission electron microscopy (HRTEM, JOEL-2010 operating at 200 kv), combined with selected-area argon-ion-beam thinning technique., Results: In the body of the lesion, the preferential core dissolution was found in most of enamel nano-crystals, whereas the peripheral dissolution of individual crystal could be occasionally observed. The initial carious dissolution of individual enamel apatite crystal occurred as a number of small electron-lucent spots along the central dark line with blurry, bent or disconnected lattice fringes. These small electron-lucent spots fused with each other to form large electron-lucent areas. Finally the central perforation was frequently seen in the crystals. The CDL always appeared in the same place with central perforation in carious crystals, which could be seen to extend along the CDL., Conclusions: The initial carious dissolution is directly related to the lattice defects in the enamel nano-crystals. The preferential core dissolution can be partly ascribed to the CDL, which is presumed to be particularly susceptible area to caries.

Objective: To observe the expression of homeobox gene Msx-1, Msx-2 and Dlx-2 during murine mandibular first molar development., Methods: The murine heads or mandibles on embryonic days 11-18 (E11-18) and postnatal day 1-3 (P1-3) were removed, fixed and embedded, 5 micro m serial sections were cut in the coronal plane. Msx-1, Msx-2 and Dlx-2 RNA probes were synthesized by in vitro transcription and labeled with digoxigenin. Msx-1, Msx-2 and Dlx-2 mRNA expression was observed after in situ hybridization., Results: During molar development Msx-1 transcripts appeared only in mesenchymal cells, not in epithelial cells. Msx-2 and Dlx-2 both expressed in the epithelial and mesenchymal cells. At the initiation stage of the molar development Msx-2 and Dlx-2 had similar expression. At the bud stage (E13-14) Msx-2 mRNA signaling was intensive in the enamel organ and slight in the dental mesenchyme; Dlx-2 signaling was stronger in the dental papilla. At cap stage (E15-16) Msx-2 showed prominent mRNA signaling in enamel knot and Dlx-2 was maximal in the dental papilla. At the late bell stage (P2-3) Msx-2 transcripts were observed in odontoblasts but not labeled in ameloblasts, and Dlx-2 transcripts appeared in ameloblasts but no labeling was seen in odontoblasts., Conclusions: Msx-1, Msx-2 and Dlx-2 are expressed in various patterns during murine mandibular first molar development, suggesting they possibly play a role in the interaction between the epithelium and mesenchyme during the molar development.

Objective: To study the distribution and expression of fibromodulin in normal periodontium, so as to understand its role in periodontal tissue homeostasis., Methods: Five normal male Lewis rats were killed and their bilateral mandibular first molars with surrounding alveolar bones and gingival tissues were taken out. Human gingival fibroblasts, fibroblasts of periodontal ligament, and osteoblasts were cultured. Immunohistochemistry with anti-fibromodulin, anti-decorin, anti-biglycan, anti-type I collagen, and anti-type III collagen antibodies and RT-PCR were used to detect the tissue distribution and cellular localization of fibromodulin and related proteoglycans, decorin and biglycan, and type I and type III collagens., Results: Fibromodulin was strongly expressed in the suprabasal gingival epithelium, periodontal ligament, alveolar bone, gingival and periodontal fibroblasts as well as their matrices. Strong expression was noted in the area close to oral gingival aspect and the interfaces of periodontal ligament-alveolar bone and periodontal-ligament-cementum. Decorin was strongly expressed in the area close to the gingival sulcus in gingival tissue. Biglycan was stained evidently in gingival epithelium. Fibromodulin, decorin and biglycan mRNAs were strongly expressed in osteoblasts. mRNAs of type I and type III collagens were strongly expressed in gingival fibroblasts., Conclusion: Fibromodulin may interact with other small proteoglycans to regulate the network formation of periodontal collagen fibers, and may be involved in mineralization of the alveolar bone and cementum.

Objective: To construct and detect the targeted anti-caries fusion DNA vaccine pGJA-P encoding the A-P fragment of pac, glu fragment of gtfB and extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) gene for the targeting antigen to APC., Methods: The extracellular region of the human CTLA4 and the Fc region of human Iggamma(1) genes were amplified by RT-PCR from human lymphocytes, and the genes were cloned into pUC(m-T) vector respectively. After sequencing, Fc region of human Iggamma(1) gene was cloned to the downstream of CTLA4 gene fragment as the recombinant plasmid pGJ. The fusion gene was then inserted into the plasmid pGLUA-P to get the recombinant plasmid pGJA-P. The CHO cells were transfected with liposome and the expression of fusion protein in cultured supernatants were detected using Western blotting., Results: The plasmids pGJ and pGJA-P carried the CTLA4-Ig fusion gene and CTLA4-Ig fusion gene, A-P fragment of pac gene and glu fragment of gtfB gene respectively. The expressed protein could response to specific anti-PAc antibody., Conclusion: The targeted fusion anti-caries DNA vaccine pGJA-P is constructed successfully and expressed in eukaryotic cells correctly.

Objective: To investigate the different characteristics of periodontal ligament cells and gingival fibroblasts in synthesizing Collagen I and III., Methods: Periodontal ligament cells and gingival fibroblasts were cultured from the same patient and detected for expression of Collagen I and III by immunocytochemical methods., Results: The immunocytochemical staining showed Collagen I and III were positive in periodontal ligament cells, and mild positive in gingival fibroblasts. Statistical analysis suggested the difference between gingival fibroblasts and periodontal ligament cells., Conclusion: There were differences between the periodontal ligament cells and gingival fibroblasts in synthesizing or secreting of Collagen matrix.

Objective: To assess the efficacy of ultrasonic irrigation of the root canal in vivo and in vitro., Methods: Sixty anterior teeth or premolars with the diagnosis of periapical periodontitis were divided into two groups (syringe irrigation group, group S and ultrasonic irrigation group, group U) and were evaluated clinically. Sixty extracted teeth with single straight canals were selected. Forty of them were divided into two groups and were evaluated histologically. Another twenty teeth were divided into two groups and were evaluated by SEM., Results: Clinically, the number of lateral canals obturated in group U was more than that in group S (P < 0.01). The incidence of pain during irrigation were 13.3% in group S, and 3.3% in group U (P > 0.05). Histologically, the amount of organic debris of the root canals in group U were significantly less than that in group S (P < 0.01). By SEM study, the number of visible open dentinal tubules in group U were significantly greater than that in group S (P < 0.05)., Conclusion: Ultrasonic irrigation of the root canal will be a useful technique in root canal therapy.

Objective: To establish a simple and rapid method to detect Streptococcus mutans and streptococcus sobrinus simultaneously in human saliva., Methods: Chromosomal DNA from the bacteria was obtained by the extraction method with phenol-chloroform. A nested PCR method with two sets of primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus), was optimized to detect S. mutans and S. sobrinus from standard strains, clinical strains and directly in human saliva., Results: The first process of nested PCR was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 10(5) CFU cells of standard and clinical strains, or from 1 ml clinical saliva samples containing 10(5) CFU cells of either species. a second process of nested PCR, using the first PCR product as a template with new internal primers to detect 10(3) CFU of either streptococcal species in 1ml saliva samples., Conclusion: Nested PCR could detect S. mutans and S. sobrinus rapidly and simply in human saliva. This finding would be important to studies of elucidation the role of these two streptococcal species in the etiology of dental caries.