Effect of root canal sealers on biocompatibility of human periodontal ligament cells / 北京大学学报(医学版)

Objective:To compare the effects of three root canal sealers with respect to time on biocom-patibility of human periodontal ligament cells (hPDLCs).The sealers included zinc oxide and eugenol based sealers (ZOE),epoxy resin sealers (ERS)and silicone based sealers (SBS).Methods:hPDLCs were primarily cultured,with the method combining of tissue explant and enzymatic digestion.The cells were then exposed to different extract fluids:(1)ZOE extracted for 24 h group ;(2)ZOE extracted for 1 week group;(3)ZOE extracted for 2 weeks group;(4)ERS extracted after 24 h group;(5)ERS extracted after 1 week group;(6)ERS extracted after 2 weeks group;(7)SBS extracted after 24 h group;(8)SBS extracted after 1 week group;(9)SBS extracted after 2 weeks group;(10)Dulbecco modified Eagle’s me-dium/F12(DMEM/F12)as negative control group.Cell morphology was observed under an inverted mi-croscope.Cell proliferation was measured by methyl-thiazol-diphenyltetrazolium (MTT)assay.ALP assay kit was used for measuring alkaline phosphatase (ALP)activity.Sealers of 2 weeks’setting time were then immersed in an osteogenic medium for examination of mineral nodules and calcium deposits.Re-sults:Considering the relative growth rate (RGR),ZOE was severely to moderately cytotoxic (RGR:13.6% -39.9%),while ERS was slightly or not cytotoxic (RGR:87.6% -95.3%).Only SBS did not show any cytotoxicity after setting (RGR:91.8% -106.7%).The setting time influenced the cytotoxic-ity of ERS which decreased after 1 week.Considering the ALP activity,there was no difference between SBS group and control group(F =3.397,P =0.053).According to the results of calcium deposits, ZOE:D562 nm =0.180 ±0.050,ERS:D562 nm =2.968 ±0.201,SBS:D562 nm =3.623 ±0.039,Control:D562 nm =3.477 ±0.102,the ranking of ALP activity and calcium deposits was as follows:ZOE

Strategies for combating bacterial biofilm infections.

Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in the hostile environment. Under the protection of biofilm, microbial cells in biofilm become tolerant and resistant to antibiotics and the immune responses, which increases the difficulties for the clinical treatment of biofilm infections. Clinical and laboratory investigations demonstrated a perspicuous correlation between biofilm infection and medical foreign bodies or indwelling devices. Clinical observations and experimental studies indicated clearly that antibiotic treatment alone is in most cases insufficient to eradicate biofilm infections. Therefore, to effectively treat biofilm infections with currently available antibiotics and evaluate the outcomes become important and urgent for clinicians. The review summarizes the latest progress in treatment of clinical biofilm infections and scientific investigations, discusses the diagnosis and treatment of different biofilm infections and introduces the promising laboratory progress, which may contribute to prevention or cure of biofilm infections. We conclude that, an efficient treatment of biofilm infections needs a well-established multidisciplinary collaboration, which includes removal of the infected foreign bodies, selection of biofilm-active, sensitive and well-penetrating antibiotics, systemic or topical antibiotic administration in high dosage and combinations, and administration of anti-quorum sensing or biofilm dispersal agents.

Strategies for combating bacterial biofilm infections / 国际口腔科学杂志·英文版

Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in the hostile environment. Under the protection of biofilm, microbial cells in biofilm become tolerant and resistant to antibiotics and the immune responses, which increases the difficulties for the clinical treatment of biofilm infections. Clinical and laboratory investigations demonstrated a perspicuous correlation between biofilm infection and medical foreign bodies or indwelling devices. Clinical observations and experimental studies indicated clearly that antibiotic treatment alone is in most cases insufficient to eradicate biofilm infections. Therefore, to effectively treat biofilm infections with currently available antibiotics and evaluate the outcomes become important and urgent for clinicians. The review summarizes the latest progress in treatment of clinical biofilm infections and scientific investigations, discusses the diagnosis and treatment of different biofilm infections and introduces the promising laboratory progress, which may contribute to prevention or cure of biofilm infections. We conclude that, an efficient treatment of biofilm infections needs a well-established multidisciplinary collaboration, which includes removal of the infected foreign bodies, selection of biofilm-active, sensitive and well-penetrating antibiotics, systemic or topical antibiotic administration in high dosage and combinations, and administration of anti-quorum sensing or biofilm dispersal agents.

Biological effect of inhibition of Notch signaling pathway on human dental pulp cells / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Notch signaling pathway on human dental pulp cells.</p><p><b>METHODS</b>The γ-secretase inhibitor N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester(DAPT) was applied to inhibit the Notch signaling pathway of human dental pulp cells. The solvent dimethly sulfoxide (DMSO) served as the negative control.Senescence conditions were evaluated by cells morphology changes, the alkaline phosphatase (ALP) expression and its activity, senescence-associated β-galactosidase (SA-β-Gal) expression and the senescence related gene p53 expression.</p><p><b>RESULTS</b>After inhibition of the Notch signaling pathway, morphology changes, including flatter cells, larger plasma area, were seen in the 10th passage human dental pulp cells. ALP expression and activity showed a significant decrease at the 8th passage after inhibition (35.36 ± 2.55) U/g, compared with the negative control group[(49.76 ± 4.30) U/g] (t = 4.989, P = 0.008).SA-β-Gal-positive cells could be seen as early as the 8th passage and more positive cells were evident at the 10th passage. The relative expression level of p53 gene was elevated in the 10th passage cells (1.7 ± 0.4) compared with the negative control group(1.0) (t = 3.581, P = 0.012).</p><p><b>CONCLUSIONS</b>Human dental pulp cells became senescent at earlier passages after inhibition of Notch signaling pathway.Notch signaling pathway may affect life cycle of human dental pulp cells.</p>

Genetic diversity and mRNA expression of F-ATPase subunit uncG gene of streptococcus mutans clinical isolates / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the genetic diversity and the gene expression of membrane-bound proton-translocating ATPase (F-ATPase) subunit gene uncG derived from Streptococcus mutans (S. mutans) clinical isolates.</p><p><b>METHODS</b>38 S. mutans strains derived from caries-active and caries-free individuals including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncG was amplified with specific primers from S. mutans genomic DNA, then the PCR product was analyzed by RFLP and sequenced. The relative expression quantity of uncG gene against the housekeeping gene recA was determined by using RT-PCR method. A gel documentation system and QUANTITY ONES software were used to analyze the data results.</p><p><b>RESULTS</b>It was testified that four genotypes A, B, C and D of PCR-RFLP were revealed when respectively digested with Alu I and Bsr I, but the distributions of the four genotype strains showed no difference (P > 0.05). The differences of uncG gene transcript quantities derived from different genotype or different aciduranc strains had no significance (P > 0.05).</p><p><b>CONCLUSION</b>This study indicated that uncG gene of F-ATPase obviously displayed genetic diversity and existed polymorphism at mRNA expression level, while the Alu I-RFLP genotypes and the expression levels would not be responsive to different acid tolerance of S. mutans strains.</p>

Genetic diversity of F-ATPase subunits gene uncEBF amplified from Streptococcus mutans clinical isolates / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this research was to study the genetic diversity of F-ATPase subunit gene uncEBF derived from Streptococcus mutans (S. mutans) clinical isolates, furthermore to investigate the relationship between the genetic diversity of F-ATPase and S. mutans aciduric ability.</p><p><b>METHODS</b>38 S. mutans strains included 18 high acid tolerance strains and 20 low acid tolerance strains. Gene uncEBF of these isolates were amplified with specific primers from S. mutans genomic DNA, and the PCR products were analyzed by RFLP and sequenced. SPSS 11.0 statistic software assayed the results.</p><p><b>RESULTS</b>It was testified that two genotypes A and B of PCR-RFLP were revealed when digested with Alu I and Dde I digested fragments of uncEBF displayed two different patterns C and D. Fisher exact two-tail test showed that the distributions of A and B genotype strains with different acidurance were different (P < 0.05), and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I and Dde I recognized sites.</p><p><b>CONCLUSION</b>This study indicated that uncEBF gene of S. mutans F-ATPase obviously exhibited genetic diversity.</p>

Genetic diversity of F-ATPase subunits gene uncA amplified from Streptococcus mutans clinical isolates / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity.</p><p><b>METHODS</b>Sixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced.</p><p><b>RESULTS</b>Two genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites.</p><p><b>CONCLUSIONS</b>This study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.</p>

Homology analysis of the extended-V region of the surface proteins in different serotype Streptococcus mutans / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To analysis the homology among the extended-V region of the surface proteins in different serotype Streptococcus mutans (c, f, d, g) and to find out it's significance in anti-caries vaccine.</p><p><b>METHODS</b>The DNA of the bacteria (standarded serotype c, d, f, g and partial serotype c clinicals) was extracted and the extended-V region (SrV+, 1 384-2 514 bp) was amplified using polymerase chain reaction (PCR). Then the products were assessed using restriction fragment length polymorphism (RFLP) by endonuclease Dde I. The genotypings were sequenced and analysised using the program of BLAST on NCBI Gene Bank database.</p><p><b>RESULTS</b>About 1.13 kb fragments were produced both in serotype c and f, the serotype d and g were failed. The RFLP results showed that five different patterns(A, B, C, D, E) among the 117 PCR products were reveled by Dde I. The ration of the genotypings A and B were the most among the strains, the C was lower, the D and E respectively was 1 and 3 strains per genotype. OMZ175 (serotype f) was belong to B genotype. Selected one of the A, B, C genotypings to sequenced and blasted. Then the results of the blastn showed that the identities of the gene sequence were 92%-98% between the serotype c and serotype f, part sequence of the serotype g was homology with the SrV+ of the serotype c, the protein sequence among serotype c, d, f, g were 77%-82%.</p><p><b>CONCLUSION</b>It is reasonable to use some putative pipetides to study the anti-caries vaccine among the extended-V regions of the surface proteins in different serotype (c, d, f, g) in S. mutans.</p>

Study on lactate dehydrogenase activity of Streptococcus mutans isolates derived from caries-active and caries-free individuals / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To preliminarily investigate the relationship between the Lactate dehydrogenase (LDH) activity of Streptococcus mutans (S. mutans) and dental caries initiation.</p><p><b>METHODS</b>100 S. mutans strains derived from caries-active and caries-free individuals were cultured in BHI medium supplemented with glucose. Cells were extracted and ruptured, and the extracted liquid protein was quantified with Coomassie brilliant blue G250 staining methods. LDH activity was assayed using the pyruvate-dependent oxidation of NADH-with and without FDP.</p><p><b>RESULTS</b>LDH activity of the two groups strains had no difference (P > 0.05), but the distribution of differ class enzyme activity strains in the two groups was different (P < 0.05).</p><p><b>CONCLUSION</b>LDH activity of S. mutans is correlated to the initiation of dental caries to some extent. The measurement methods in this study can be applied in preliminary quantitation of LDH activity and the screening of S. mutans strains.</p>

Detection of the transmitted strains and non-transmitted strains of Mutans streptococci by AP-PCR / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Dental caries is a transmissible infectious disease in which S. mutans plays the major role. The purpose of this study was to detect the S. mutans transmitted strains and non-transmitted strains by AP-PCR fingerprint for laying the foundation of study on the relation between bacterial properties of S. mutans and its transmission.</p><p><b>METHODS</b>Plaque samples were obtained from buccal surfaces of 20 3-4 years old children and their mothers. Caries experience was scored by dmft (DM-FT). Diet regime, breast feeding and care of the children were recorded. 200 Isolates of S. mutans were detected by S. mutans B medium and confirmed biochemically. DNA from each isolate was purified and AP-PCR fingerprinting was conducted. Amplicons were separated by electrophoresis in 1.5% agarose gels.</p><p><b>RESULTS</b>45 different patterns among the 200 isolates were found. There were 10 mothers (50%) and 15(75%) children owning one genotype while 10 mothers and 5 children owning more than one (2 mothers owning 5 types). The data showed that the mothers harbored a more heterogeneous population of S. mutans than their children. Comparisons in genotypes between children and their mothers discovered that 16(80%) children harbored the same genotypes as their mothers, indicating high transmission in the group of people. Detection of the S. mutans transmitted strains and non-transmitted strains in mothers demonstrated that 10 (50%) mothers harbored not only transmitted strains but also non-transmitted strains, suggesting that different strains had different ability of transmission.</p><p><b>CONCLUSION</b>AP-PCR was capable of detecting the S. mutans transmitted strains and non-transmitted strains. Some S. mutans genotypes had higher ability of transmission than others.</p>

A study on screening effective immunization route of anticaries DNA vaccine pcDNA3-gtfB / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Glucosyltransferase-B (GTF-B) of Streptococcus mutans has been implicated as a principal virulent factor in the development of dental caries. The objective was to use recombined plasmid pcDNA-gtfB expressing multiple antigen of glucosyltransferase-B as gene vaccine to immunize rats through different route, and to investigate the immunization effects of immunization routes.</p><p><b>METHODS</b>A total of 18 Wistar rats were divided into 3 groups, including the quadriceps injection group, the intransal irrigation group and the submandibular gland-targeted injection group. The serum IgG and salivary IgA were assayed by using ELISA after pcDNA3-gtfB immunization. The serum IgG and salivary IgA in different groups were compared using statistical one-way ANOVA.</p><p><b>RESULTS</b>Compared these 3 groups, the serum IgG in the quadriceps injection group was much higher than those of other two groups (P < 0.01), while the salivary IgA of the submandibular gland-targeted injection was much higher than those of other two groups (P < 0.01).</p><p><b>CONCLUSION</b>It is indicated pcDNA3-gtfB is good candidate for anticarious gene vaccine, and submandibular gland-targeted injection is an effective immunization route for stimulating salivary IgA.</p>

Relationship between genetic diversity of surface protein of Streptococcus mutans and dental caries.Ⅱ Sequence of V-region and P-region gene of surface protein of Streptococcus mutans / 实用口腔医学杂志

Objective: To sequence V-region and P-region gene of surface protein of serotype c Streptococcus mutans clinical isolates with different adherent abilities. Methods: The clinical isolates of serotype c S.mutans included two groups with different spaP-pv AluI genotypes, which were derived from previous studies in our lab. The genome DNA was extracted. The spaP-pv (2 060-3 157 bp) was amplified by PCR, then sequenced and analyzed. Results: Seven sites of AluI 5′-AG↓CT- 3′were included in the sequences of spaP-pv of both genotypes strains. The sequences of spaP-pv of ten a-genotype strains were the same except several site mutations, and so were those of b-genotype strains. Two different DNA fragments were revealed between a and b geotype strains. And they were located in the V-region of spaP. Conclusion: The mutation of gene encoding V-region of S.mutans clinical isolates might be related to the differences of adherent abilities .

PAGE-AgNO_3 staining displays AP-PCR fingerprint of mutans streptococci / 实用口腔医学杂志

Objective:To investigate whether the AP-PCR fingerprint of mutans streptococci(MS) can be displayed by PAGE-AgNO 3 staining. Methods: Amplification products of 200 MS clinical isolates by AP-PCR was separated and stained by ?=3.5%PAGE-AgNO 3 and agarose-EB respectively. Results were compared and the agreement value of Kappa between two methods was calculated. Results: ?=3.5%PAGE-AgNO 3 discerned both homogeneity and heterogeneity of MS genotypes, just as agarose-EB,Kappa value for agreement was 1.00 . Moreover, more bands was showed by PAGE-AgNO 3 staining than by agarose-EB, so PAGE-AgNO 3 gave a clearer pattern than agarose-EB. Conclusion: AP-PCR fingerprint of MS can be displayed by ?=3.5%PAGE-AgNO 3 staining.