Micro-tensile bond strengths to sclerotic dentin using a self-etching and a total-etching technique.

OBJECTIVES: To evaluate the in vitro regional micro-tensile bond strengths of a self-etching/self-priming adhesive system to sclerotic dentin, in the absence or presence of phosphoric acid conditioning. METHODS: Naturally-occurring, non-carious cervical lesions on extracted premolars were hand-cleaned with a slurry of pumice and chlorhexidine, then bonded without further cavity preparation. One group was bonded using Clearfil Liner Bond 2V (Kuraray Co. Ltd., Osaka, Japan). The other group was first conditioned with K-etchant; 40% phosphoric acid gel (Kuraray) prior to the application of the self-etching primer. Artificially prepared wedge-shaped cavities were also made in sound premolars and bonded with the two methods as controls. Resin composite build-ups were made using Clearfil Protect Liner F and AP-X resin composite (Kuraray). After storage in water for 24h, the teeth were sectioned into 0.7 x 0.7 mm composite-dentin beams along the occlusal and gingival walls, and at the apex of the lesions. The use of two conditioning methods, two substrate types, and three different locations yielded 10-14 beams for each of the 12 groups. After testing for the microTBS, representative beams that were stressed to failure were examined with SEM. Remaining fractured beams were demineralized and processed for TEM examination. Statistical analysis was performed using a three-way ANOVA and Student-Newman-Keuls tests. RESULTS: Regardless of the conditioning methods, bond strengths to sound dentin were significantly higher than to sclerotic dentin (P< 0.05). With sclerotic dentin, there was no significant difference for the conditioning methods used, except that K-etchant significantly improved the bond strength at the gingival aspect of the lesions. Fractographic analysis revealed that the self-etching primer could not etch beyond the surface hypermineralized layer of sclerotic dentin. Interfacial failure occurred along the surface of the mineralized intermicrobial matrix and/or hypermineralized layer. With the use of phosphoric acid, a hybrid layer was only seen when the surface layers were thin. Incompletely removed sclerotic casts were evident in both groups. SIGNIFICANCE: Removal of the surface layers of sclerotic dentin and/or conditioning with stronger acids may be beneficial to obtain stronger bonding to sclerotic dentin.

Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS.

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5'-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.

An ultrastructural study of the application of dentine adhesives to acid-conditioned sclerotic dentine.

OBJECTIVE: This in vitro study examined the ultrastructure of resin-infiltrated sclerotic dentine following the application of a self-etching primer, with or without the adjunctive use of phosphoric acid pre-conditioning. MATERIALS AND METHODS: Non-carious, natural cervical sclerotic lesions were hand-cleaned with a slurry of pumice and chlorhexidine and bonded without further cavity preparation. One group was bonded using Clearfil Liner Bond 2V (Kuraray Co. Ltd, Osaka, Japan) alone. Specimens from the other group were pre-conditioned with K-etchant (40% phosphoric acid gel, Kuraray) prior to the application of the same self-etching primer. Artificially prepared wedge-shaped lesions were also made in sound bicuspids, bonded using the two methods, and used as controls for the two groups. For SEM examination, each specimen was cryofractured into two halves through a pre-formed slit made on the lingual surface, after the respective conditioning treatment. Different locations within the lesions were examined after rinsing of the phosphoric acid/self-etching primer and specimen dehydration. For TEM investigation, specimens were bonded with the adhesive. Both demineralised and undemineralised ultrathin sections were prepared from the occlusal, gingival and deepest part of the wedge-shaped bonded lesions following specimen fixation, dehydration and resin embedding. RESULTS: A hypermineralised surface layer was present on the surface of etched sclerotic dentine. This layer was thicker in the deepest part of the natural lesions, where bacterial colonisation of the lesion surface was also apparent. Both treatment protocols were unable to effectively dissolve sclerotic casts that occluded the dentinal tubules. Depending upon the thickness of the surface layers at different locations in the natural lesion, self-etching primer treatment alone resulted in reduction of the thickness of the authentic hybrid layer (i.e. hybridised intertubular dentine). This was also true of phosphoric acid pre-conditioning along the deepest part of the natural lesions. Within this region, intertubular dentine completely devoid of an authentic hybrid layer could be seen in both treatment groups. Resin tags were also sparsely observed in such regions. CONCLUSIONS: Adhesive strategies that rely mostly on micromechanical retention alone may be compromised by the sporadic absence of the hybrid layer and resin tags in sclerotic dentine. Based on the ultrastructural features presented, it is further speculated that adaptive strategies such as removal of the surface layers and extended etching time may not be completely effective in improving bonding efficacy in highly sclerotic dentine. Interdisciplinary research should be continued to develop alternative procedures for bonding resins equally well to sound and sclerotic dentine.

Bonding of a self-etching primer to non-carious cervical sclerotic dentin: interfacial ultrastructure and microtensile bond strength evaluation.

PURPOSE: The objectives of this study were 1) to examine the ultrastructural features of the resin-sclerotic dentin interface following the application of Clearfil Liner Bond II sigma to natural cervical wedge-shaped lesions, and 2) to evaluate the regional tensile bond strength of this self-etching primer at different locations on natural and artificially-created cervical lesions. MATERIALS AND METHODS: Deep cervical natural lesions were bonded using the self-etching primer. Micromorphology of the bonded interface at different locations within the lesions were examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning transmission electron microscopy/energy dispersive x-ray analysis (STEM/EDX). Ultrastructural features were further compared with the use of the same self-etching primer on artificial lesions created in sound cervical dentin. A nontrimming technique was used to evaluate the regional tensile bond strength from the occlusal, gingival, and the deepest central part of both natural and artificial cervical lesions. Beams with a mean area of 0.46 +/- 0.03 mm2 were prepared and were pulled to failure using a Bencor Multi-T testing device attached to an Instron universal tester. Bond strength results were evaluated using a two-way ANOVA design. RESULTS: A hypermineralized layer devoid of intact, banded collagen was invariably present on the surface of the natural lesions. Depending upon its thickness at different locations of the lesion, the action of a self-etching primer may be limited to this surface layer alone, producing a hybridized hypermineralized surface layer. Penetration of the self-etching primer into the underlying sclerotic dentin produced a hybridized complex containing a hybridized hypermineralized surface layer as well as a subsurface layer of hybridized intertubular dentin. Bacterial colonization of the lesion surface resulted in the formation of an additional zone of hybridized intermicrobial matrix over the surface of the lesions. Dentinal tubules remained blocked with sclerotic casts, and resin tags were rarely observed. Regional tensile bond strength results showed that the overall bond strength to natural sclerotic dentin was about 20% lower than sound cervical dentin, but was independent of the different locations within the lesions from which bond strength was evaluated. CONCLUSION: There were four factors that may have influenced the overall decrease in bond strength in natural cervical sclerotic lesions: a) the presence of a hybridized intermicrobial matrix together with entrapped bacteria may have weakened the bonds, b) inability of a self-etching primer to etch through a thick, hypermineralized surface layer, c) presence of a layer of possibly remineralized, denatured collagen at the base of the hypermineralized surface layer, and d) retention of acid-resistant sclerotic casts that obliterate the tubular lumina and prevent effective resin tag formation.

Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

Characterization of the Pac25I restriction-modification genes isolated from the endogenous pRA2 plasmid of Pseudomonas alcaligenes NCIB 9867.

Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-modification (R-M) system, Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of the Pac25I methylase revealed significant similarity with the XcyI, XmaI, Cfr9I, and SmaI methylases. High sequence similarity was displayed between the Pac25I endonuclease and the XcyI, XmaI, and Cfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5'-C CCGGG-3') and are thus perfect isoschizomers. However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5'-CCCGGG-3'). Both the Pac25I methylase and endonuclease were expressed in Escherichia coli. An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of the Pac25I R-M operon. In addition, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.

Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes.

Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.

Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region.

The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.