[Research progress in ecological prevention of dental caries].

Dental caries is one of the most common oral diseases around the world. Dental plaque attached to the surfaces of teeth is the main biological factor leading to caries. Although fluoride is still one of the most commonly used methods to prevent caries, with the change of epidemiological characteristics of caries and the update of the understanding of caries etiology, it is necessary to use other ecological methods such as antimicrobial peptides, arginine, probiotics and natural products, etc. to enhance the effect of fluoride in preventing dental caries. The present article reviews the research progress on the ecological approaches for caries prevention in recent years.

[An analysis of key points for root canal therapy technique].

The success rate of root canal therapy(RCT)have been improved continuously along with the advancement in RCT techniques in the past several decades. If standard procedures of modern RCT techniques are strictly followed, the success rate of RCT may exceed 90%. The success of RCT is mainly affected by such factors as clear concept of the anatomy of root canals, proper mechanical and chemical preparation and perfect filling of root canal system. If these factors are sufficiently noted, a success is easy to achieve. Even though the primary RCT fails, retreatment can further be conducted to save the diseased teeth.

Immortalization and characterization of human dental papilla cells with odontoblastic differentiation.

AIM: To establish a cell line of immortalized human dental papilla cells (hDPCs). METHODOLOGY: Primary hDPCs were cultured and infected with lentivirus containing the hTERT gene. Integration and transcription of the hTERT gene were verified by PCR. The characteristics of the cells, such as morphology, proliferation and mineralization, were analysed. Also, the expression of odontoblastic-related markers including ALP, DMP1, DLX3, OSX, DSP and Nestin, was detected by immunohistochemistry and real-time RT-PCR. RESULTS: hTERT gene was integrated into genomic DNA of immortalized cells (hDPC-TERT) and transcribed into mRNA. With long-time culture, hDPC-TERT bypassed senescence and grew over 120 population doublings. hDPC-TERT cells have a higher proliferation rate, but retain the phenotypic characteristics of the primary hDPCs, and so was ALP activity and mineralization activity. Furthermore, the hDPC-TERT cells express no DSP and Nestin with maintenance medium, but highly expressed DSP and Nestin after odontoblastic induction. CONCLUSIONS: A line of immortalized human dental papilla cells, which remains in an undifferentiated state and has odontoblastic differentiation potential, was established. This cell line can be used as a cell model for studying the mechanism of the initiation of odontoblast differentiation.

Flagellin-PAc fusion protein is a high-efficacy anti-caries mucosal vaccine.

We previously demonstrated that an anti-caries DNA vaccine intranasally administered with recombinant flagellin protein as a mucosal adjuvant enhanced salivary IgA response and conferred better protection against caries. However, the relatively weak immunogenicity of DNA vaccines and the necessity for a large quantity of antigens remain significant challenges. Here, we fused the flagellin derived from E. coli (KF) and target antigen PAc containing the A-P fragment of PAc from S. mutans (rPAc) to produce a single recombinant protein (KF-rPAc). The abilities of KF-rPAc to induce rPAc-specific mucosal and systemic responses and protective efficiency against caries following intranasal immunization were compared with those of rPAc alone or a mixture of rPAc and KF (KF + rPAc) in rats. Results showed that KF-rPAc promoted significantly higher rPAc-specific antibodies in serum as well as in saliva than did an equivalent dose of rPAc alone or a mixture of KF + rPAc. Intranasal immunization of 8.5 µg KF-rPAc could achieve 64.2% reduction of dental caries in rats. In conclusion, our study demonstrated that flagellin and PAc fusion strategy is promising for anti-caries vaccine development, and KF-rPAc could be used as an anti-caries mucosal vaccine.

Validation of micro-CT against the section method regarding the assessment of marginal leakage of sealants.

BACKGROUND: The aim of this study was to validate the micro-CT and related software against the section method using the stereomicroscope for marginal leakage assessment along the sealant-enamel interface. METHODS: Pits and fissures of the occlusal surface of 10 teeth were sealed with a resin-fissure sealant material without acid etching, thermocycled for 5000 cycles, immersed in 50% silver nitrate for three hours and scanned using micro-CT. Teeth were embedded in epoxy resin and cut in three sections. The middle section was subjected to micro-CT and stereomicroscopy. Images were taken from the left and right sides of the sealant-enamel interface at both the left and the right site of the section. Two experienced evaluators assessed marginal leakage. RESULTS: Both assessment instruments observed no leakage in 37 out of the 40 images evaluated. Leakage at the sealant-enamel interface was observed in three stereomicroscopy images only. A fracture line in the sealant was seen on eight stereomicroscopy images and observed in only two micro-CT images. CONCLUSIONS: The quality of the micro-CT and related software used in the present study does not qualify it to replace the section method as the gold standard for marginal leakage assessment at the sealant-enamel interface of permanent teeth.

Caries-preventive effect of sealants produced with altered glass-ionomer materials, after 2 years.

OBJECTIVES: The aim of the present study was to investigate the caries-preventive effect of sealants produced with a high-viscosity glass-ionomer with an elevated powder-liquid ratio (ART), of having energy added to this glass-ionomer, and that of glass-carbomer, in comparison to that of resin composite sealants. METHODS: The randomized controlled trial covered 407 children, with a mean age of 8 years. At a school compound three dentists placed sealants in pits and fissures of high caries-risk children. Evaluation by two independent evaluators was conducted after 0.5, 1 and 2 years. The Kaplan-Meier survival method, ANOVA and t-test were used in analyzing the data. RESULTS: 1352 first permanent molars were sealed. 6.6% of children and 6.8% of sealants dropped out within 2 years. 27 re-exposed pits and fissures, 20 in occlusal and 7 in smooth surfaces, in 25 children, developed a dentin carious lesion. The cumulative survival of dentin carious lesion-free pits and fissures in the glass-carbomer sealant group was statistically significantly lower (97.4%) than those in the high-viscosity glass-ionomer with energy supplied (99%) and the resin-composite (98.9%) sealant groups. There was no statistically significant difference in the cumulative survival of dentin carious lesion-free pits and fissures, between the high-viscosity glass-ionomer with (99%) and without (98.3%) energy supplied sealant groups, after 2 years. SIGNIFICANCE: The survival of dentin carious lesion-free pits and fissures was high in all sealant types. More dentin carious lesions were observed in the glass-carbomer sealant group.

Flagellin enhances saliva IgA response and protection of anti-caries DNA vaccine.

We and others have shown that anti-caries DNA vaccines, including pGJA-P/VAX, are promising for preventing dental caries. However, challenges remain because of the low immunogenicity of DNA vaccines. In this study, we used recombinant flagellin protein derived from Salmonella (FliC) as a mucosal adjuvant for anti-caries DNA vaccine (pGJA-P/VAX) and analyzed the effects of FliC protein on the serum PAc-specific IgG and saliva PAc-specific IgA antibody responses, the colonization of Streptococcus mutans (S. mutans) on rat teeth, and the formation of caries lesions. Our results showed that FliC promoted the production of PAc-specific IgG in serum and secretory IgA (S-IgA) in saliva of rats by intranasal immunization with pGJA-P/VAX plus FliC. Furthermore, we found that enhanced PAc-specific IgA responses in saliva were associated with the inhibition of S. mutans colonization of tooth surfaces and endowed better protection with significant fewer caries lesions. In conclusion, our study demonstrates that recombinant FliC could enhance specific IgA responses in saliva and protective ability of pGJA-P/VAX, providing an effective mucosal adjuvant candidate for intranasal immunization of an anti-caries DNA vaccine.

Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats.

Early life vaccination is necessary to protect young children from dental caries. Our group had previously reported that a plasmid DNA vaccine pGJA-P/VAX against the glucosyltransferase (GTF) enzyme and cell surface antigen AgI/II (PAc) of Streptococcus mutans (S. mutans) elicited a specific and protective immunity in adult experimental animal models. In this report, early life immunization with the same plasmid was studied following intranasal (i.n.) and intramuscular (i.m.) delivery in murine models. The potential of inducing mucosal and systemic immune responses to special antigens was measured by ELISA. In addition, cytokine production and protection effectiveness against dental caries formation were also investigated. In the i.n. route, rats were primed when they were 5 days old, and boosted after 10 and 20 days with either plasmid pGJA-P/VAX-bupivacaine complexes, or pGJA-P/VAX alone, or empty vector. The pGJA-P/VAX-bupivacaine combination was able to mount the immune responses characterized by increased antibody levels of specific salivary IgA and serum IgG, preferential IFN-gamma production and significant reduction in the dental caries lesions. In the i.m. route, rats were vaccinated with either pGJA-P/VAX alone or empty vector with the same immunization schedule as the i.n. route. Plasmid pGJA-P/VAX alone induced a significant increase in the serum IgG and IFN-gamma production. However, it was not effective in eliciting specific salivary IgA and in decreasing the dental caries formation. All these findings indicate the feasibility of immunity with a targeted fusion DNA vaccine to a young immune system.

Fusion protein encoded by a CTLA-4 targeted DNA construct binds to human dendritic cells.

Fusing antigens to cytotoxic T-lymphocyte antigen 4 (CTLA-4) represents an effective approach to enhance DNA vaccine efficacy. It has been speculated that the direct targeting of CTLA-4 fusion antigens to antigen-presenting cells (APCs) causes antigens to be processed and presented to T cells more efficiently, leading to a stronger immune response. In the present study, dendritic cells (DCs), the most potent APCs, were generated from human monocytes. The specific binding of CTLA-4 fusion protein to DCs was investigated by flow cytometry. The results showed that the CTLA-4 fusion protein was capable of binding to the B7 molecules on human DCs with specificity.

Dentin phosphoprotein frameshift mutations in hereditary dentin disorders and their variation patterns in normal human population.

BACKGROUND: Dentin phosphoprotein (DPP) is the most abundant non-collagenous protein in dentin, which is highly phosphorylated and plays key roles in dentin biomineralization. The aetiology of isolated hereditary dentin disorders in most affected families is largely unknown and the association between DPP and dentin disorders has not been well established. This study aims to determine whether there are some involvements for DPP mutations in inherited dentin disorders and to clarify the sequence variation patterns of DPP in normal population. METHODS: Genomic DNA was analysed in eight families with hereditary dentin disorders and 110 individuals in the normal population. The full coding sequence of DPP was amplified by polymerase chain reaction (PCR) and screened for mutations and variations by direct sequencing and TOPO TA-cloning sequencing. RESULTS: Five frameshift mutations in DPP coding region were identified in five of the eight families. The mutations co-segregated with the disease phenotypes in affected families and were not found in 220 control chromosomes. In the normal population, we revealed 14 in-frame indels (insertion/deletion), six non-synonymous single nucleotide polymorphisms (SNPs), and five synonymous SNPs in the DPP coding region. These variants display extensive linkage disequilibrium and constitute a total of 15 haplotypes with three predominant haplotypes in the investigated normal population. CONCLUSIONS: Our data provide the first evidence that DPP mutations can cause hereditary dentin disorders and suggest that in-frame length variations and missense SNPs in DPP have no obvious pathogenetic effects on dentin formation.

Mesenchyme is responsible for tooth suppression in the mouse lower diastema.

Between the incisor and molars in each dental quadrant, mice have a toothless gap (diastema) that may contain vestigial tooth primordia. It is still not clear whether suppression of odontogenesis in the mouse lower diastema can be attributed to epithelium, mesenchyme, or both. Therefore, using recombination experiments with mouse tissues from E11.5 and E13.5 stages, we investigated whether the epithelium or mesenchyme is responsible for the suppression of odontogenesis. Five groups of recombinants were established and cultured under mouse kidney capsules. The results demonstrated that at E11.5, the lower diastemal epithelium and mesenchyme possessed odontogenic potential and competence, respectively; at E13.5, both the lower diastemal epithelium and mesenchyme had odontogenic competence, while the lower diastemal mesenchyme did not possess odontogenic potential. On the basis of comparison of the odontogenic capabilities between the lower diastemal and molar tooth primordia, we conclude that mesenchyme is responsible for tooth regression in the mouse lower diastema.

Protective efficacy of a targeted anti-caries DNA plasmid against cariogenic bacteria infections.

We have previously reported that a targeted anti-caries DNA plasmid pGJA-P/VAX which was constructed against the antigenic determinants of Streptococcus mutans (S. mutans) successfully induced antibody responses in mice and monkeys. The present study explored the protective efficacy of pGJA-P/VAX against cariogenic bacterial challenge. Groups of rats were orally challenged with S. mutans or Streptococcus sobrinus (S. sobrinus) and then immunized with pGJA-P/VAX or the vector pVAX1 intranasally. Serum IgG and salivary IgA antibody levels were assessed by an enzyme-linked immunosorbent assay and caries activity was evaluated by the Keyes method. The results showed that specific salivary IgA antibody responses were induced following intranasal vaccination with pGJA-P/VAX. Moreover, immunization with pGJA-P/VAX resulted in significantly reduced enamel and dentinal caries lesions in rats after S. mutans infection and significantly reduced enamel caries lesions after S. sobrinus infection. Thus, pGJA-P/VAX was not only protective toward S. mutans infection, but also provided cross-strain protection against S. sobrinus infection in rats.

Immunogenicity and persistence of a targeted anti-caries DNA vaccine.

We have previously reported that a targeted anti-caries DNA vaccine, pGJA-P, induced accelerated and increased antibody responses compared with a non-targeted anti-caries DNA vaccine. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Here, we report on our exploration of the kinetics of the antibody responses generated following pGJA-P/VAX immunization and the persistence of pGJA-P/VAX at both the inoculation site and the draining lymph nodes. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for more than 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization.

The inhibitory effect of magnolol from Magnolia officinalis on glucosyltransferase.

Dental caries has been an intractable disease in spite of intense dental research. Glucosyltransferase (GTF) enzyme plays the most important role in the development of dental caries. In our previous studies, magnolol, a compound from Magnolia officinalis Rehder et Wilson (Magnoliaceae), was shown to possess a strong anti-GTF activity. The purpose of the present study was to examine the effect of magnolol on the functional domains of GTF for the purpose of defining its anti-GTF activity mechanism. GTF-I which was prepared from Streptococcus milleri transformant KSB8 cells expressing the gtfB gene was used. The results demonstrated magnolol reduced total glucan synthesis, depending on the magnolol concentration. There were no significant differences in Michaelis constant (K(m)) values between the presence and absence of magnolol as determined by Lineweaver-Burk plot, and maximum velocity (V(m)) in the presence of magnolol was lower than that in its absence. Magnolol significantly inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Free glucose in the presence of magnolol was reduced by 33-48% as compared to in its absence, while the quantity of glucan was reduced by 75-82%. These findings suggested that magnolol inhibited both two sequential reaction phases of GTF non-competitively by operating on the glucan-binding domain, but not on the catalytic domain. Magnolol could be a valuable resource for the exploration of novel bioactive compounds in natural products.

A two-year randomized clinical trial of chlorhexidine varnish on dental caries in Chinese preschool children.

The objective of this study was to assess the effect of six-monthly professional applications of chlorhexidine varnish on the prevention of dental caries in primary molars in Chinese preschool children. In a double-blinded, randomized, placebo-controlled clinical trial, 334 children aged 4-5 years were randomly divided into two groups. Children in the test group received six-monthly applications of a 40% chlorhexidine varnish, and the control children received a placebo varnish. Caries status of the children was assessed by two calibrated examiners at baseline and after 24 months, according to criteria recommended by the World Health Organization. The two-year mean caries increments in the test and the control group children were 1.0 and 1.6 decayed, missing, or filled molar surfaces (dmfs-molar), respectively, a 37.3% reduction (t test, p = 0.036). No side-effects were found. It was concluded that six-monthly applications of chlorhexidine varnish were effective in reducing the incidence of dental caries in primary molars.

Evaluation of the accuracy of three electronic apex locators using glass tubules.

AIM: To evaluate the accuracy of three different electronic apex locators (EALs) using glass tubules. METHODOLOGY: Forty-eight glass tubules with different diameters and an agar model were used to mimic root canals. A size 15 stainless steel K-file was used as the measuring electrode. The Root ZX, Propex and Neosono Ultima EZ were used to measure the tubule length with tubules dry, or filled with 0.9% NaCl, 3% H(2)O(2), 2.5% NaOCl or 17% EDTA. The distance between the real length (RL) and measured length (ML) of the tubules was recorded. The range of RL +/- 0.5 mm and RL +/- 1 mm was used to evaluate the accuracy of the EALs. Results were subject to correlation analysis and Friedman's test. RESULTS: In dry tubules, the accuracy of Root ZX was 75-91.7% for RL +/- 0.5 mm and 100% for RL +/- 1 mm, whilst the measurements of the other two EALs were all within the RL +/- 0.5 mm. No influence from the increase in tubule diameter on the accuracy of all three EALs was observed in dry tubules. In tubules filled with electrolyte, the accuracy of the Root ZX decreased as tubule diameter increased (R(d) > 0, P < 0.05). The RL-ML distance recorded by Propex was inversely related to the tubule diameter (R(d) < 0, P < 0.05). The accuracy of Propex was 75-100% for RL +/- 0.5 mm and 100% for RL +/- 0.5 mm when the tubule diameter was not more than 0.80 mm, but decreased in tubules with diameter over 0.80 mm and filled with 2.5% NaOCl or 17% EDTA. Nearly, all the measurements (except for six tubules) using Neosono Ultima EZ were within 1 mm shorter than RL despite the contents in tubules and the increase of tubule diameter. CONCLUSIONS: The accuracy of the Root ZX decreased as the tubule diameter increased when tubules were filled with electrolytes. The electrolytes in the tubules decreased the accuracy of Propex when the tubule diameter was large. The electrolytes in tubules and tubule diameter had no influence on the accuracy of Neosono Ultima EZ. The Propex and Neosono Ultima EZ were more accurate than the Root ZX under various conditions in this laboratory study.

The aggressive form of cherubism: report of two cases in unrelated families.

Cherubism is a benign lesion that causes painless symmetrical enlargement of the jaws, usually with a familial tendency. We describe in two Chinese families two cases of the aggressive form of cherubism with extensive swelling on both sides of the mandible, typical microscopic findings, and apparent familial history.

Differentiation ability of rat postnatal dental pulp cells in vitro.

The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immunochemistry by stem cell marker STRO-1 proved the existence of stem cells or progenitors in the isolated cell population. The dissociated cells were then cultured both on smooth surfaces and on three-dimensional (3-D) scaffold materials in medium supplemented with beta-glycerophosphate, dexamethasone, and L-ascorbic acid. Cultures were analyzed by light and scanning electron microscopy and, on proliferation, alkaline phosphatase activity and calcium content were determined and the polymerase chain reaction was performed for dentin sialophosphoprotein, osteocalcin, and collagen type I. These cells showed the ability to differentiate into odontoblast-like cells and produced calcified nodules, which had components similar to dentin. In addition, we found that the "odontogenic" properties of the isolated cells were supported by three-dimensional calcium phosphate and titanium scaffolds equally well.

A comparison of instrument-centering ability within the root canal for three contemporary instrumentation techniques.

AIM: To study and compare Great Taper (GT) hand files using a reversed balanced force technique, nickel-titanium (NiTi)flex files with a balanced force technique and stainless steel (SS) K-type files using a step-back technique. METHODOLOGY: Forty-eight extracted mandibular premolar teeth with single root canals having curvatures between 15 and 45 degrees were prepared using a modified Bramante model and randomly divided into three groups. The teeth were cross-sectioned at 2, 6 and 10 mm from the working length. Preoperative images of canals at three levels were captured at 20x magnification using a stereomicroscope. Canals in each group were, respectively, prepared to an apical size.10 GT file with 0.2 mm tip diameter, size 30 NiTiflex file and size 30 SS K-file. The GT file was used in a reversed balanced force technique, the NiTiflex file was used in a balanced force technique, and the SS K-file was used in a step-back technique. Postoperative canals were imaged under the conditions same as those for the preoperative canals. The postoperative images were superimposed over the preoperative images using software photoshop 6.0. The ability to maintain the instrument in the central axis of the canal and the deviation from the central canal axis were determined and compared by statistical analysis, along with the assessment of the amount of dentine removed. RESULTS: At apical level, the centering ratio, the distance of transportation and the dentine removed in GT and NiTiflex groups were significantly less than those in SS group (P < 0.01), but no statistical differences were found between the two NiTi groups. At other levels, there were no substantial differences amongst the groups. CONCLUSIONS: Compared with SS K-files, GT hand files and NiTiflex files remain better centered and produce significantly less transportation in curved canals.

Construction and immunogenic characterization of a fusion anti-caries DNA vaccine against PAc and glucosyltransferase I of Streptococcus mutans.

Glucosyltransferases (GTFs) and A cell-surface protein (PAc) are two important virulence factors of the cariogenic organism Streptococcus mutans. They may mediate sucrose-independent or sucrose-dependent attachment of Streptococcus mutans to tooth surfaces, respectively. Thus, inhibiting both virulence factors is predicted to provide better protection against caries than inhibiting a single factor. To develop a highly efficient vaccine against caries, we constructed a fusion DNA vaccine, pGLUA-P, by cloning the GLU region of GTF into a DNA vaccine, pCIA-P, which encodes two highly conservative regions of PAc. In this report, we provide evidence that fewer caries lesions were observed in rats following subcutaneous injection of pGLUA-P, compared with pCIA-P, near the submandibular gland. Our findings suggest that a multigenic DNA vaccine may be more caries-preventive than a single-gene DNA vaccine.