Antibiofilm potential over time of a tricalcium silicate material and its association with sodium diclofenac.

OBJECTIVES: The objectives of this study are to investigate, over time, the antimicrobial activity against polymicrobial biofilms and ability to inhibit biofilm formation, of Biodentine (BD) alone and with 5% and 10% sodium diclofenac (DC). MATERIAL AND METHODS: The antimicrobial activity of BD alone and modified with 5% and 10% DC against polymicrobial biofilm growth in dentin was determined by a modified direct contact test. The study groups were (1) BD; (2) BD + 5% DC; and (3) BD + 10% DC. The viability of microorganisms after 1 and 4 weeks was quantified by means of an ATP assay and flow cytometry. The antibiofilm efficacy of the materials, preventing polymicrobial biofilm formation over time, was assessed by confocal laser scanning microscopy (CLSM). RESULTS: The results obtained with both the ATP test and flow cytometry showed that BD alone and with 5% and 10% DC exerted antibiofilm activity with respect to the control, in the two evaluated times (p < 0.001). Comparison between groups showed a tendency of increased antimicrobial effect, both over time and depending on the DC concentration. These results coincide with those obtained in CLSM analysis, where efficacy increased with time and DC concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Biodentine, over time, showed antimicrobial and antibiofilm efficacy on polymicrobial biofilms. The addition of 5% and 10% DC to BD enhanced this effect, in a concentration- and time-dependent manner.

Influence of dentine debris and organic tissue on the properties of sodium hypochlorite solutions.

AIM: To determine the free available chlorine of 2.5% sodium hypochlorite (NaOCl) alone and combined with 9% etidronic acid (HEDP) in the presence of inhibitors, organic tissue and organic tissue plus dentine debris; to evaluate the influence of dentine debris on the tissue-dissolving capacity of both NaOCl solutions; and to determine the antimicrobial action of these solutions when in contact with organic tissue and organic tissue plus dentine debris. METHODOLOGY: The available chlorine of the solutions over time in the absence and presence of the inhibitors was measured using a titration method. The organic tissue dissolution by the solutions alone and in the presence of dentine powder was evaluated by weighing bovine tissue specimens before and after exposure to the solutions for 3 and 10 min. For the antimicrobial activity, biofilms of Enterococcus faecalis were exposed to the solutions for 3 min in the absence and presence of organic tissue and organic tissue + dentine debris. The biovolume and percentage of damaged membrane cells of the biofilm were measured by means of confocal microscopy and the live/dead technique. Nonparametric tests were used to determine statistical differences (P < 0.05). RESULTS: Both inhibitors consumed the free available chlorine of the solutions over time. The presence of dentine debris significantly reduced the tissue dissolution capacity of the NaOCl solutions (P < 0.05). The percentages of biovolume reduction were not affected by the presence of the inhibitors in the two NaOCl solutions, whereas the percentage of damaged membrane cells was significantly reduced (P < 0.001). Overall, a similar behaviour was observed in the NaOCl and NaOCl/HEDP groups. CONCLUSIONS: The presence of organic tissue and organic tissue + dentine debris favoured rapid consumption of the free chlorine of NaOCl and NaOCl/HEDP. This resulted in a decreased ability to dissolve organic tissue without affecting the short-term antimicrobial activity.

Efficacy of antimicrobial solutions against polymicrobial root canal biofilm.

AIM: To evaluate the antibiofilm activity of 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), 2% alexidine (ALX) and 0.2% cetrimide (CTR) alone and in combination on mature polymicrobial root canal biofilms on human dentine using confocal laser scanning microscopy (CLSM). METHODOLOGY: Twenty-eight human dentine specimens were infected for 21 days with microbial samples collected from infected root canals of three volunteers. Antibiofilm activity of the irrigating solutions was evaluated after 3 min of contact time under CLSM. For quantification purposes, bioimage_L software was used. The variables evaluated were the log10 of total biovolume (µm3 ) and percentage of live cells (green) population. Statistical analysis of both variables was performed using an anova test and a post hoc Duncan test to determine significant clusters amongst groups. The variable green population percentage was previously subjected to the normalized Anscombe transformation. RESULTS: The NaOCl group had a total biovolume and percentage of live cells significantly lower than the other groups (P Ë‚ 0.001). The addition of 0.2% CTR significantly increased the antimicrobial effect of 2% CHX (P Ë‚ 0.001). There were no significant differences between 0.2% CTR, 2% ALX and the combination of both (P Ë‚ 0.05). CONCLUSIONS: Overall, 2.5% NaOCl dissolved and killed bacteria significantly more efficiently when used against polymicrobial mature biofilm on human dentine. Cetrimide improved the antimicrobial activity of chlorhexidine and alexidine.

The effect of benzalkonium chloride additions to AH Plus sealer. Antimicrobial, physical and chemical properties.

OBJECTIVES: The aim of this study was to determine the antimicrobial and antibiofilm activities and physicochemical properties of AH Plus sealer mixed with different concentrations of benzalkonium chloride (BC). METHODS: AH Plus was tested alone and mixed with 1%, 2% and 3% of BC. The antimicrobial and antibiofilm activities of the sealers against Enterococcus faecalis were evaluated by the direct contact test (DCT) and by confocal laser scanning microscopy, respectively. Setting time, flow and solubility were assessed according to ANSI/ADA specifications. Microhardness and contact angle tests were also performed. The chemical changes of the sealers were evaluated by X-ray diffraction analysis, and both Fourier transform infrared spectroscopy (FT-IR) and attenuated total reflectance Fourier transform infrared (ATR FT-IR). RESULTS: AH Plus+3% BC was the only sealer to promote total elimination of E. faecalis and the biovolume in this group was significantly lower than in the rest of the sealers (p>0.05). The physical properties of the sealers were according to the ANSI/ADA specifications. The microhardness decreased significantly when BC was added and a significant reduction in contact angle was obtained when incorporating 2% and 3% BC (p<0.05). No phase changes were observed with the modified sealers. CONCLUSIONS: The addition of 2% or higher concentrations BC to AH Plus showed antimicrobial and antibiofilm activities without affecting the properties specified in ANSI/ADA standards. However, additives to the root canal sealer altered other physical and chemical properties that are not commonly found in the literature to evaluate filling materials. CLINICAL SIGNIFICANCE: The present study highlights that the antimicrobial properties of AH Plus can be significantly improved with the addition of BC. Testing beyond what is specified in standards may be indicated.

Antimicrobial activity of Chlorhexidine, Peracetic acid and Sodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms.

AIM: To evaluate the antimicrobial effect of 2.5% sodium hypochlorite alone (NaOCl) and associated with 9% HEBP (NaOCl/HEBP), 2% peracetic acid (PAA) and 2% chlorhexidine (CHX), on the viability of Enterococcus faecalis biofilms attached to dentine. METHODOLOGY: Biofilms of E. faecalis were grown on the surface of dentine blocks for 5 days and then exposed to the irrigating solutions for 3 min. Distilled water was used as the control. The total biovolume and the percentage of dead cells of the infected dentine were measured by means of confocal microscopy and the live/dead technique. Nonparametric tests were used to determine statistical differences (P < 0.05). RESULTS: NaOCl and the NaOCl/HEBP mixture were associated with a significantly greater percentage of dead cells, followed by PAA (P < 0.05). No significant antimicrobial effect of CHX was observed in comparison with the control group. Total biovolume decreased significantly in NaOCl, NaOCl/HEBP and PAA solutions in comparison with the CHX and control groups. CONCLUSIONS: NaOCl alone or associated with HEBP were the most effective irrigant solutions in dissolving and killing E. faecalis biofilms.

Reduction in Enteroccocus faecalis counts - a comparison between rotary and reciprocating systems.

AIM: To compare the chemomechanical reduction and regrowth of Enterococcus faecalis between rotary and reciprocating systems in root canal preparation. METHODOLOGY: Seventy-six single-rooted human mandibular premolars were selected and standardized to 15 mm in length. Root canals were enlarged up to a size 25 K-file and irrigated with distilled water and then were infected with E. faecalis for 4 weeks. The specimens were divided into 3 groups (n = 24) for instrumentation with Mtwo, Twisted File and WaveOne. Each group was further divided into two subgroups (n = 12) according to the irrigant used: distilled water or 5.25% sodium hypochlorite (NaOCl). Before and after rotary preparation, microbiological samples were collected using three sterilized paper points, and efficacy was expressed as reduction in percentage. The proportion of grown samples for 60 days was evaluated using nonparametric Kaplan-Meier survival analysis. Differences amongst groups were tested using the log-rank test at a significance level of 0.05. RESULTS: In the main root canal, the percentage reduction in the distilled water and 5.25% NaOCl groups ranged from 95.9% to 100%, with no significant differences amongst the three systems (P > 0.05). The bacterial regrowth in NaOCl groups revealed that Mtwo had the lowest number of samples regrown at 60 days, giving statistically significant differences with respect to Twisted File (P = 0.029) and WaveOne (P = 0.005). CONCLUSIONS: Reciprocating and rotary systems resulted in similar percentage reduction in E. faecalis when using either distilled water or 5.25% NaOCl solution. Over time, the Mtwo system was more effective regarding disinfection.

Dissolving efficacy of eucalyptus and orange oil, xylol and chloroform solvents on different root canal sealers.

AIM: To evaluate the solubility of five root canal sealers in orange oil, eucalyptol, xylol and chloroform solvents. METHODOLOGY: The solubility of RoekoSeal, Sealer 26, Epiphany, Endomethasone and EZ-Fill sealers was assessed in orange oil, eucalyptol, xylol, chloroform and distilled water. Seventy-five samples of root canal sealers were prepared and then divided into five groups for immersion in solvent for 2, 5 or 10 min. The means of loss weight were determined for each material in each solvent at all immersion periods, and the values were compared by factorial analysis of variance (anova) and SNK multiple comparisons. RESULTS: In the orange and eucalyptus oil groups, there was no significant difference among RoekoSeal, Sealer26, Epiphany and EZ-Fill at the three immersion periods (P > 0.05). With xylol, no significant differences were found at 5 and 10 min (P > 0.05) for each root sealer. Orange and eucalyptus oil solvents were as effective as chloroform at 2 min in dissolving all the root sealers. CONCLUSIONS: Xylol was the most effective solvent followed by the chloroform and the essential oils (eucalyptol and orange oil). Orange oil behaved in a similar way to eucalyptus oil.

Topographical evaluation of the major apical foramen in permanent human teeth.

AIM: To determine the distance from the anatomical root apex to the major apical foramen and the position of the major foramen on the root apex. METHODOLOGY: Crowns of 926 human teeth were sectioned at the cementum-enamel junction. Specimens were mounted on microscope slides for measurement parallel to the long axis of the teeth. The major foramen was identified as the largest-diameter opening at the root apex. A total of 1331 root specimens were evaluated using an optical stereomicroscope to an accuracy of 0.01 mm at 40 x (+/-10) magnification. The distance from the anatomical apex to the most apical point of the major foramen was measured, and its location (central, buccal, lingual, mesial and distal) was recorded. RESULTS: The mean distance between the major foramen and the anatomical root apex was 0.69 mm; the mean distance was larger in posterior teeth (0.82 mm) and smaller in anterior teeth (0.39 mm). A wide range of anatomical apex to major foramen distances were observed in all tooth groups: the greatest distance was in maxillary molars (0.95 mm) followed by mandibular pre-molars (0.87 mm) and mandibular molars (0.80 mm). The major foramen was at the tip of the root in 40% of teeth. The most frequent deviations of the foramen were to the buccal (20%) and distal (14%). CONCLUSION: In this sample of teeth without apical resorption the distance between the major foramen and the anatomical root apex was always <1 mm. Deviation of the major foramen from the anatomic apex varied widely amongst tooth groups.

Decalcifying effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine.

AIM: To evaluate and compare ex vivo the decalcifying effect of 15% EDTA, 15% citric acid, 5% phosphoric acid and 2.5% sodium hypochlorite on root canal dentine. METHODOLOGY: Two 2-mm-thick slices were cut from the coronal third of the root of 10 human incisors. Each slice was sectioned into two equal parts. Specimens were assigned to one of four groups (n = 10) for immersion in 20 mL of either 15% EDTA, or 15% citric acid, 5% phosphoric acid or 2.5% NaOCl, for three time periods (5, 10 and 15 min). The concentration of Ca(2+) extracted from the dentine was measured by atomic absorption spectrophometry. The amount of calcium extracted was analysed using the Kruskal-Wallis test for global comparisons and the Mann-Whitney U-test for pairwise comparisons. RESULTS: In the three time periods, 15% EDTA and 15% citric acid extracted the largest amount of calcium, with no significant differences between them. The 2.5% NaOCl solution extracted insignificant amounts of calcium, whereas 15% EDTA extracted 86.72% of the calcium in the first 5 min, and 15% citric acid and 5% phosphoric acid had a similar pattern of calcium removal (77.03% and 67.08% in first 5 min, respectively). CONCLUSIONS: Solutions of 15% EDTA, 15% citric acid and 5% phosphoric acid decalcify root dentine, with most calcium extracted during the first 5 min of action. The efficacy of 15% citric acid and 15% EDTA solutions was significantly greater than that of 5% phosphoric acid solution at each time period (5, 10 and 15 min).

In vitro evaluation of the accuracy of three electronic apex locators.

The accuracy of three electronic apex locators (EALs) (Justy II, Root ZX, and Neosono Ultima EZ) is evaluated, together with the concordance of the measurements obtained by two different operators. Twenty single-root human teeth were used, sectioning the crown to gain access to the root canal. A first operator (A) determined the reference (or control) length (corresponding to the actual length) for each tooth, after which all teeth were measured individually and independently by the other two operators (B and C). The results obtained with each EAL and by each operator were in turn compared with the corresponding control length. The statistical analysis of the results showed EAL reliability in detecting the apex to vary from 80% to 85% and 85% to 90% (depending on the operator) for the Justy II and Neosono systems, respectively, whereas reliability was found to be 85% for the Root ZX device. These results, combined with a high interobserver concordance, suggest electronic root canal measurement to be an objective and acceptably reproducible technique.

A comparative study of apical leakage of Endomethasone, Top Seal, and Roeko Seal sealer cements.

A comparison was made of the apical leakage of three sealers. Fifty single-root human teeth were randomly divided into 5 groups (n = 10; 3 experimental and 2 control). The teeth of the positive-control and experimental groups were instrumented with K-type files to size 45. The experimental groups were obturated by laterally-vertically, condensed gutta-percha with Endomethasone, Top Seal, or RSA sealer cements. The positive-control group was nonobturated and the negative-control group was noninstrumented. The root surfaces were then coated with nail varnish (except the apex in the experimental groups) and immersed in black ink (for 1 week at 37 degrees C). The statistical evaluation of the results obtained by clearing and cross-section techniques showed no significant differences between sealers. Leakage, as determined by the clearing technique, was significantly greater than that quantified by cross-section analysis.

Influence of time and thermocycling on marginal sealing of several dentin adhesive systems.

This study evaluated the in vitro microleakage of six dentin adhesive systems. Triangle-shaped Class V cavities with coronal margin in enamel and gingival margin in cementum or root dentin were cut in the buccal surfaces of 90 non-carious single-root human teeth. These teeth were randomly assigned into six groups (n = 15) for the evaluation of six different dentin adhesive systems: One Step, Prime & Bond 2.0, Syntac Single, Single Bond, Optibond Solo and Syntac Sprint. The preparations were restored with Degufill Ultra composite and polished using the Enhance system. Each group was randomly divided into three subgroups (n = 5): samples of the first subgroup were immersed in 2% methylene blue solution for seven days; those of the second subgroup remained in a similar solution for 31 days; those of the third subgroup were thermocycled 500x at 5-55 degrees C and immersed in 2% methylene blue for seven days. All 90 teeth were then embedded in methacrylate and bucco-lingually sectioned; the dye penetration was evaluated using an 0-4 ordinal scale. All of the dentin adhesive groups showed minimal leakage at the enamel margins with increased leakage at the gingival margins. Optibond Solo showed the best outcomes among the dentin adhesives tested.

4.1R proteins associate with interphase microtubules in human T cells: a 4.1R constitutive region is involved in tubulin binding.

Red blood cell protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles and partners of specific nonerythroid 4.1R isoforms, we analyzed 4.1R in human T cells and found that endogenous 4.1R was distributed to the microtubule network. Transfection experiments of T cell 4.1R cDNAs in conjunction with confocal microscopy analysis revealed the colocalization of exogenous 4.1R isoforms with the tubulin skeleton. Biochemical analyses using Taxol (paclitaxel)-polymerized microtubules from stably transfected T cells confirmed the association of the exogenous 4.1R proteins with microtubules. Consistent with this, endogenous 4.1R immunoreactive proteins were also detected in the microtubule-containing fraction. In vitro binding assays using glutathione S-transferase-4.1R fusion proteins showed that a constitutive domain of the 4.1R molecule, one that is therefore present in all 4.1R isoforms, is responsible for the association with tubulin. A 22-amino acid sequence comprised in this domain and containing heptad repeats of leucine residues was essential for tubulin binding. Furthermore, ectopic expression of 4.1R in COS-7 cells provoked microtubule disorganization. Our results suggest an involvement of 4.1R in interphase microtubule architecture and support the hypothesis that some 4.1R functional activities are cell type-regulated.

A constitutive region is responsible for nuclear targeting of 4.1R: modulation by alternative sequences results in differential intracellular localization.

Red blood cell protein 4.1, 4.1R, is an extreme variation on the theme of isoform multiplicity. The diverse 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, are localized at different intracellular sites, including the nucleus. To characterize nonerythroid 4.1 proteins lacking the most upstream translation initiation site, analyze their intracellular localization and define specific domains involved in differential intracellular targeting of 4.1R, we cloned 4.1 cDNAs lacking that translation initiation site. Seven different 4.1R cDNAs were isolated. Four of these encoded 4.1R proteins localized predominantly to the nucleus and the other three localized to the cytoplasm. Three of the nuclear 4.1R isoforms did not contain the nuclear localization signal previously identified in the alternative exon 16. A comparative analysis of the exon composition of the naturally occurring 4.1R cDNAs cloned and of appropriate composite cDNA constructs, with the subcellular distribution of their respective products, demonstrated that a region encoded by constitutive exons, which is therefore common to all 4.1R isoforms and has been termed 'core region', had the capacity of localizing to the nucleus. This region was able to confer nuclear targeting to a cytosolic reporter. In protein 4.1R isoforms, the nuclear targeting of the core region is modulated by the expression of alternative exons. Thus, exon 5-encoded sequences eclipsed nuclear entry of the core region, resulting in 4.1R isoforms that predominantly distributed to the cytoplasm. Exon 5 was also able to confer cytoplasmic localization to a nuclear reporter. In protein 4.1R isoforms, when exons 5 and 16 were both expressed the nuclear targeting effect of exon 16 was dominant to the inhibitory effect observed by the expression of exon 5, yielding proteins that predominantly localized to the nucleus. Taken together, these results indicate that all 4.1R molecules contain a conserved region that is sufficient to target the protein to the nucleus, but that specific exon-encoded sequences modulate this capacity by acting in a hierarchical order.

The N-terminal 209-aa domain of high molecular-weight 4.1R isoforms abrogates 4.1R targeting to the nucleus.

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase-PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of approximately 145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

Study of the shear bond strength of five one-component adhesives under simulated pulpal pressure.

Recently, several adhesive systems have been introduced that combine the primer and bonding resin in a single bottle. The purpose of this study was to evaluate the bonding efficiency of these one-component adhesives under conditions of simulated pulpal pressure and to determine the influence of storage time on the shear bond strength. One hundred caries-free human molars were embedded with epoxy resin in cylindrical rubber molds. Flat dentin surfaces at a level 1 mm above the pulpal chamber were obtained and used as the region for bonding. The specimens were randomly assigned to five groups (n = 20): (1) Syntac Single, (2) Prime & Bond 2.0, (3) One Step, (4) Single Bond, and (5) OptiBond Solo. Each bonding system was combined with the same composite resin (Herculite XRV). After resin polymerization, half of the samples from each group were tested at 1 week and the other half at 4 weeks. During the bonding procedure and storage time a pulpal pressure of 20 cm of serum was applied. Analysis of the data by one-way ANOVA testing showed that the shear bond strengths were significantly different (P < 0.001). OptiBond Solo and Single Bond presented the best results. As the storage time increased there was a significant decrease in the shear bond strength for all the adhesive systems used.

An alternative domain determines nuclear localization in multifunctional protein 4.1.

Multiple protein 4.1 isoforms are originated by alternative pre-mRNA splicing, differential use of two translation initiation sites, and posttranslational modifications. The complexity of alternative splicing events suffered by the 4.1 pre-mRNA makes necessary the direct cloning of 4.1 full-coding cDNA sequences to ensure that the encoded 4.1 proteins are naturally occurring isoforms. We have approached this point by reverse transcription-polymerase chain reaction techniques using RNA from the nucleated human Molt-4 T-cell line as a starting template. Molecular cloning of 4.1 cDNAs using the second translation initiation codon has allowed us to identify two 4.1 isoforms, designated 4.1H and 4.1I, which are differentially targeted to the nucleus (4.1H) and the cytoplasm (4.1I). These two isoforms differ only in the inclusion (4.1H) or exclusion (4.1I) of 21 amino acids encoded by exon 16. A cluster of basic amino acids, KKKR, generated by joining of the sequences encoded by the constitutive exon 13 and the alternative exon 16, is necessary for the nuclear targeting of 4.1H, as demonstrated by site-directed mutagenesis analysis. Immunofluorescence microscopy and biochemical studies indicate that 4.1H belongs to the group of nuclear 4.1 proteins that are distributed diffusely throughout the nucleoplasm and that are extractable in 0.5% Triton X-100. This is the first demonstration of differential nuclear targeting by the presence of an alternative domain, among naturally occurring protein 4.1 isoforms.

[The area of dentinal diffusion at the time of the manual preparation of root canals]. / Aire de diffusion dentinaire lors de la préparation manuelle des conduits radiculaires.

In this current piece of work we study the area of dentinal diffusion obtained with various irrigating agents which eliminate the smear layer: critic acid at concentrations of 10, 25 and 50%, 15% ethylene-diamino-tetraacetic acid (EDTA), and REDTA (compound of EDTA). All the agents proved their efficiency in eliminating the smear layer, which was corroborated by use of the sweep electron microscope and objectified using computerized image analysis, thus obtaining different diffusion areas with the various irrigating solutions used.

[Mechanical instrumentation of the root canals. A study using SEM and computerized image analysis]. / Instrumentation mécanique des conduits radiculaires. Une étude à l'aide du M.E.B. et analyse informatisée d'image.

The effectiveness of ultrasonic and sonic instrumentation in eliminating the smear layer from instrumented root channels. The results were examined with S.E.M. and the effective areas of dental diffusion were calculated using the computerized image analyzer. The agents used: citric acid at concentrations of 10, 25 and 50% as well as 15% EDTA, proved their efficiency with both types of mechanical instrumentation. However, the area of diffusion found was always greater using the ultrasonic instrumentation technique as opposed to the sonic instrumentation technique. 1, 2.5 and 5.25% sodium hypochlorite, as well as 10 volume hydrogen peroxide were not effective in eliminating the smear layer using both types of instrumentation.