The aim of this systematic review was to appraise the diagnostic accuracy of signs/symptoms and tests used to determine the condition of the pulp in teeth affected by deep caries, trauma or other types of injury. Radiographic methods were not included. The electronic literature search included the databases PubMed, EMBASE, The Cochrane Central Register of Controlled Trials and Cochrane Reviews from January 1950 to June 2011. The complete search strategy is given in an Appendix S1 (available online as Supporting Information). In addition, hand searches were made. Two reviewers independently assessed abstracts and full-text articles. An article was read in full text if at least one of the two reviewers considered an abstract to be potentially relevant. Altogether, 155 articles were read in full text. Of these, 18 studies fulfilled pre-specified inclusion criteria. The quality of included articles was assessed using the QUADAS tool. Based on studies of high or moderate quality, the quality of evidence of each diagnostic method/test was rated in four levels according to GRADE. No study reached high quality; two were of moderate quality. The overall evidence was insufficient to assess the value of toothache or abnormal reaction to heat/cold stimulation for determining the pulp condition. The same applies to methods for establishing pulp status, including electric or thermal pulp testing, or methods for measuring pulpal blood circulation. In general, there are major shortcomings in the design, conduct and reporting of studies in this domain of dental research.

Aim  To investigate dental pulp healing after tooth replantation in rats using nestin as an odontoblastic marker for immunohistochemical analysis.

Methodology  Twenty-five maxillary right first molars from 25 female Sprague-Dawley rats, aged 4 weeks post-natally, were extracted and immediately repositioned in the original socket within 5 s. Five rats each were later killed on days 3, 5 and weeks 1, 2 and 4. The maxillae were removed en bloc and the tissue samples containing the maxillary right first molars were decalcified, sectioned, mounted and stained with anti-nestin antibody to be observed under a light microscope.

Results  At 3 days after replantation, there was a localized inflammatory reaction, but pulp revascularization and healing had begun in the root area. At 5 days after replantation, odontoblast-like cells were observed. Reparative dentine deposition was observed beneath the pulp-dentine border from 1 week after replantation, and gradually increased until 2 weeks after replantation. The presence of odontoblast-like cells and the formation of reparative dentine continued from the first week throughout the experimental period. At week four, deposition of osteodentine and cementum-like tissues were observed.

Conclusions  Pulpal mineralization after replantation initially occurred via the deposition of reparative dentine, followed by the deposition of osteodentine and cementum-like tissues in rat teeth.

Aim  To compare root canal treatments performed before and after education in a nickel–titanium rotary technique (NiTiR) with respect to costs for instrumentation and number of instrumentation sessions in a County Public Dental Service in Sweden.

Methodology  Following education, 77% of the general dental practitioners adopted completely the NiTiR. The randomly selected sample comprised 850 root canal treatments: 425 performed after the education, mainly using the NiTiR-technique (group A) and 425 performed before, using mainly stainless steel hand instrumentation (SSI) (group B). The number of instrumentation sessions in root canal treatments in group A and B was calculated. A CMA was undertaken on the assumption that treatment outcome was identical in group A and B. Direct costs associated with SSI and NiTiR were estimated and compared. Investment costs required for implementation of NiTiR were calculated, but not included in the CMA.

Results  Instrumentation sessions were counted in 418 (98%) root canal treatments performed in group A and 419 (99%) in group B. The number of instrumentation sessions in group A was significantly lower; 2.38, compared with 2.82 in group B (P < 0.001). Thus, on average, for every second root canal treatment performed after the education, one instrumentation session was saved. Root canal treatments in teeth with one canal, and three or more canals, were completed in significantly fewer instrumentation sessions after the education (P < 0.001). Direct costs of instrumentation sessions were SEK 2587 (USD 411) for group A and SEK 2851 (USD 453) for group B, for teeth with one canal, and SEK 2946 (USD 468) for group A and SEK 3510 (USD 558) for group B, for teeth with three or more canals (year 2011). Root canal treatments of teeth with two canals showed no significant difference with respect to number of instrumentation sessions and costs.

Conclusions  Significantly fewer instrumentation sessions were required in group A, and root canal instrumentation therefore costs less than in group B. On the assumption that treatment outcome is identical in group A and B, root canal instrumentation performed after the education was more cost-effective.

Aim  To report a case of apical fenestration and overfilling in which unusual pain characteristics made differential diagnosis challenging.

Summary  A 32-year-old woman with diffuse, spontaneous, moderate pain in the maxillary left posterior sector, exacerbated by masticatory and facial muscle movement, with intense sporadic electric-shooting pain, underwent clinical examination and 3D cone beam computed tomography (CBCT). Apical fenestration with protrusion of the mesial root of tooth 26 beyond the buccal cortical plate, extrusion of canal filling material into the soft tissues and a periosteal reaction were detected. Surgery was performed under the operating microscope. The filling material and surrounding fibrous tissue were located, dissected from healthy soft tissues and removed. The mesiobuccal root apex was resected with a bur to within the bony crypt. A root end was prepared and filled with Tech Biosealer RootEnd™ (Isasan, Como, Italy). At the 2-week recall, the patient had complete resolution of the symptoms and good soft-tissue healing. The 1-year recall examination and intra-oral radiography confirmed complete resolution of the symptoms and health of periradicular tissues.

Key learning points 

• • 
  Apical fenestration may occur in 9% of cases and may be considered an anatomic predisposing factor for persistent pain after root canal treatment.
• • 
  This complication provides a considerable differential diagnostic challenge and is often misdiagnosed and mistreated.
• • 
  When correctly diagnosed through an accurate, multidisciplinary approach, it may be managed with a simple surgical procedure in which the endodontist should play a key role.
• • 
  Misdiagnosis and over treatment of apical fenestration, through the surgical management of chronic facial pain conditions, could lead to severe exacerbation of chronic pain, which may potentially become persistent or, indeed, intractable.

Aim  To highlight one of the possible complications associated with the inter-radicular placement of orthodontic miniscrews.

Summary  This case report describes the endodontic treatment and surgical repair of an iatrogenic root perforation involving a maxillary first molar tooth following the placement of an orthodontic miniscrew placed for anchorage purposes in the treatment of an adult patient. The orthodontic treatment plan was completed. The long-term follow-up shows a successful treatment outcome.

Key learning points 

• •
   Inter-radicular placement of orthodontic miniscrews is a valuable source of anchorage in the treatment of orthodontic patients.
• •
   Root perforation is a possible complication from inter-radicular placement of orthodontic miniscrews.
• •
   Root perforation can be successfully treated, but may involve apical surgery.

Aim  To design and evaluate the impact of virtual reality (VR) pre-surgical practice on the performance of actual endodontic microsurgery.

Methodology  The VR system operates on a laptop with a 1.6-GHz Intel processor and 2 GB of main memory. Volumetric cone-beam computed tomography (CBCT) data were acquired from a fresh cadaveric porcine mandible prior to endodontic microsurgery. Ten inexperienced endodontic trainees were randomized as to whether they performed endodontic microsurgery with or without virtual pre-surgical practice. The VR simulator has microinstruments to perform surgical procedures under magnification. After the initial endodontic microsurgery, all participants served as their own controls by performing another procedure with or without virtual pre-surgical practice. All procedures were videotaped and assessed by two independent observers using an endodontic competency rating scale (from 6 to 30).

Results  A significant difference was observed between the scores for endodontic microsurgery on molar teeth completed with virtual pre-surgical practice and those completed without virtual presurgical practice, median 24.5 (range = 17–28) versus median 18.75 (range = 14–26.5), P = 0.041. A significant difference was observed between the scores for osteotomy on a molar tooth completed with virtual pre-surgical practice and those completed without virtual pre-surgical practice, median 4.5 (range = 3.5–4.5) versus median 3 (range = 2–4), P = 0.042.

Conclusions  Pre-surgical practice in a virtual environment using the 3D computerized model generated from the original CBCT image data improved endodontic microsurgery performance.

Aim  To show the radiographic manifestation of sodium hypochlorite after accidental injection past the apical foramen and into the soft tissues.

Summary  A female patient was seen for an emergency visit after suffering a sodium hypochlorite accident at her general dentist’s office. The patient was seen within 1 h of the accident and was in pain associated with facial swelling. Radiographs, including a Cone Beam Computed Tomography (CBCT), and photographs were taken. Endodontic emergency treatment was initiated. The patient was reassured and given pain medication and antibiotics. Follow-up visits were scheduled over 6 days when the swelling had resolved.

Key learning points 

• • 
  Importance of multiple radiographic images during preoperative endodontic evaluation when undertaking endodontic retreatment.
• • 
  Knowledge of apical anatomy as related to surrounding structures.
• • 
  Effect of sodium hypochlorite when injected in the soft tissues.

Aim  To assess the ability of sodium hypochlorite (NaOCl) to penetrate simulated lateral canals and to reach working length (WL) when using the self-adjusting file (SAF).

Methodology  Seventy single-rooted teeth with oval-shaped canals were used. Upon access, presence of a single canal was confirmed by direct visualization under a dental-operating microscope. Canal length and patency were obtained using a size 10 K-file and root length standardized to 18 mm. Pre-enlargement was restricted to the coronal one-third. The apical size of each canal was gauged at WL and samples larger than size 30 were excluded. Canals were instrumented for 5 min using the SAF system while delivering a total of 20 mL of 5.25% NaOCl and 5 mL of 17% EDTA. Then, the apical diameters were standardized to size 35 using hand files. Four hundred and twenty simulated lateral canals were then created during the clearing process and roots coated with wax to create a closed system. All samples were then cleared and randomly assigned to four experimental groups: 1 (n = 15) positive pressure; 2 (n = 15) SAF without pecking motion; 3 (n = 15) SAF with pecking motion; 4 (n = 15) apical negative pressure (ANP) irrigation and (n = 10) control groups. Samples were scored on the basis of the ability of the contrast solution to reach WL and permeate into the simulated lateral canals to at least 50% of the total length. The Kruskal–Wallis test was used to analyse irrigant penetration and the Tukey test to determine statistical differences between groups (P < 0.05).

Results  All samples irrigated with ANP were associated with irrigant penetration to WL (Table 1). The differences between group 4 (ANP) and all other groups were significant in penetration to WL (P < 0.05). The pecking motion allowed for further penetration of the irrigant when using the SAF system but failed to irrigate at WL. None of the experimental groups demonstrated predictable irrigation of simulated lateral canals.

Conclusions  In this laboratory model, ANP was the only delivery system capable of irrigating consistently to full WL. None of the systems tested produced complete irrigation in artificial lateral canals.

Aim  To test the efficacy of a two-stage retreatment method in which the Self-Adjusting File (SAF) is used to remove root canal filling residue left in the canal after using ProTaper Universal retreatment files.

Method  One of the curved mesial canals of 25 extracted mandibular molars was instrumented up to ProTaper F2, followed by NiTi K-files up to size 45 and filled. Radiographs of these canals served as a pre-treatment control, which was later compared with those of the same canals after each stage of the retreatment procedure. Stage 1 consisted of ProTaper Universal D1–D3 retreatment files, which were used to remove the bulk of root filling material, followed by stage 2 in which a SAF file was applied to remove the remaining root canal filling residue. The amount of the radiopaque material in each third of the canal was evaluated before and after each stage, using bucco-lingual and mesio-distal radiographs. The amount of residue was expressed as the per cent of the root canal filled area, as measured in the pre-treatment control. The difference between the control and results of the first and second stages of retreatment was analysed using the Wilcoxon signed-rank test.

Results  Radiopaque residue was present after the use of the ProTaper Universal rotary files in 7.8 (±12.9)%, 12.9 (±13.9)% and 34.7 (±22.8)% of the coronal, mid-root and apical areas, respectively. Following the supplementary application of the SAF, the amounts of residue were reduced to 2.6 (±3.7)%, 4.1 (±5.7)% and 6.7 (±9.4)% of the coronal, mid-root and apical canal areas, respectively (P < 0.01 for the coronal and mid-root and P < 0.001 for the apical third).

Conclusion  No system removed the root filling materials entirely. The use of the SAF after rotary instrumentation using ProTaper Universal retreatment files resulted in a significant reduction in the amount of filling residue in curved canals of mandibular molars.

Aim  To evaluate the cyclic fatigue resistance of Reciproc^® and WaveOne^® instruments in simulated root canals.

Methodology  Two groups of 15 NiTi endodontic instruments of identical tip size of 0.25 mm were tested, group A; Reciproc^® R25 and group B: WaveOne^® primary. Cyclic fatigue testing was performed in a stainless steel artificial canal manufactured by reproducing the instrument’s size and taper. A simulated root canal with a 60° angle of curvature and 5-mm radius of curvature was constructed for both the instruments tested. The centre of the curvature was 5 mm from the tip of the instrument and the curved segment of the canal was approximately 5 mm in length. The Reciproc^® instruments were activated using the preset programme specific for the Reciproc^® instruments, whilst the WaveOne^® instruments were activated using the preset programme specific for the WaveOne^® instruments. All instruments were rotated until fracture occurred and the time to fracture (TtF) and the length of the fractured tip were recorded and registered. Means and standard deviations of TtF and fragment length were calculated for each system and data were subjected to Student’s t-test (P < 0.05).

Results  A statistically significant difference (P < 0.05) was noted between Reciproc^® and WaveOne^® instruments. Reciproc^® R25 instruments were associated with a significant increase in the mean time to fracture when compared with primary WaveOne^® instruments (130.8 ± 18.4 vs. 97.8 ± 15.9 s). There was no significant difference (P > 0.05) in the mean length of the fractured fragments between the instruments.

Conclusions  Reciproc^® instruments were associated with a significantly higher cyclic fatigue resistance than WaveOne^® instruments.

Aim  To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7.

Methodology  Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferroni’s t-test for multigroup comparisons.

Results  BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P < 0.05) during 2-h incubation period. BisGMA was found to increase TNF-α secretion in a dose-dependent manner (P < 0.05). In addition, CD11, CD14, CD45, CD54, CD40, CD80, and MHC II were significantly stimulated by BisGMA in a dose-dependent manner (P < 0.05). However, MHC I expression was not affected by BisGMA (P > 0.05).

Conclusions  Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

Aim  To determine the effect of increasing the proportion of zinc on partially stabilized cement (PSC) produced using a one-step sol gel process.

Methodology  A one-step sol-gel process of Portland cement-based PSC with Zn was synthesized by replacing iron nitrate. The crystalline phases of the PSC-Zn powder were analysed by using X-ray diffraction (XRD). The experimental groups [i.e. MTA, PSC-Fe (control), PSC with 1% Zn, PSC with 3% Zn, and PSC with 5% Zn] were immersed in simulated body fluid for 3 h, 1 and 3 days to evaluate the hydration product formation. The microstructure and surface morphology were analysed using scanning electron microscopy (SEM). Initial and final setting times of the materials were determined using an ASTM Vicat needle testing machine. To evaluate the cytotoxicity of PSC-Zn system, primary osteoblasts cell lines were used.

Results  The addition of increased weight percentages of Zn, resulted in a more unstable phase which favoured the formation of a monoclinic structure of C₃S with an increased hydration reaction of PSC and reduced setting time. The cytotoxicity testing of PSC with Zn revealed that the material was not toxic.

Conclusions  The newly synthesized PSC-Zn material had short setting time and was biocompatible.

Aim  To compare the efficacy of two rotary NiTi retreatment systems and Hedström files in removing filling material from curved root canals.

Methodology  Curved root canals of 57 extracted teeth were prepared using FlexMaster instruments and filled with gutta-percha and AH Plus. After determination of root canal curvatures and radii in two directions, the teeth were assigned to three identical groups (n = 19). The root fillings were removed with D-RaCe instruments, ProTaper Universal Retreatment instruments or Hedström files. Pre- and postoperative micro-CT imaging was used to assess the percentage of residual filling material as well as the amount of dentine removal. Working time and procedural errors were recorded. Data were analysed using analysis of covariance and analysis of variance procedures.

Results  D-RaCe instruments were significantly more effective than ProTaper Universal Retreatment instruments and Hedström files (P < 0.05). Hedström files removed significantly less dentine than the rotary NiTi systems (P < 0.0001). D-RaCe instruments were significantly faster compared to both other groups (P < 0.05). No procedural errors such as instrument fracture, blockage, ledging or perforation were detected in the Hedström group. In the ProTaper group, four instrument fractures and one lateral perforation were observed. Five instrument fractures were recorded for D-RaCe.

Conclusions  D-RaCe instruments were associated with significantly less residual filling material than ProTaper Universal Retreatment instruments and hand files. Hedström files removed significantly less dentine than both rotary NiTi systems. Retreatment with rotary NiTi systems resulted in a high incidence of procedural errors.

Aim  To evaluate morphological changes to the major foramen after overinstrumentation with ProTaper Universal and ProFile Vortex Ni–Ti rotary instruments.

Methodology  Twenty-eight mesiobuccal canals of maxillary and mandibular first molars were divided into two groups of 14 canals each. The root canals were prepared with ProTaper Universal or ProFile Vortex instruments. ProTaper and Vortex instruments were used until the file tip protruded 1 mm beyond the working length (0.5 mm beyond the major foramen). The major foramen was photographed before and after overinstrumentation with each file of the two systems used. The images were superimposed and evaluated using Adobe Photoshop. The parameters evaluated were canal transportation, centring ability and shape of the major foramen. Transportation and centring ability were calculated in two directions: the direction of maximum curvature (MC) and a direction vertical to the maximum curvature (VC). Measurements of canal transportation and centring ability were analysed by anova followed by post hoc least significance difference (LSD) multiple comparisons.

Results  No significant differences were observed amongst the different instruments with respect to centring ability in either direction (P > 0.05). The F3 ProTaper Universal instrument was associated with a higher mean values for transportation in the direction of MC (P < 0.05) than the S1, S2 and F1 ProTaper Universal instruments and the size 15, 0.06 taper, size 20, 0.06 taper, and size 25, 0.06 taper ProFile Vortex instruments. The size 30, 0.06 taper ProFile Vortex instrument had a larger mean value for transportation in the direction of MC (P < 0.05) than the S1 ProTaper Universal and size 15, 0.06 taper ProFile Vortex instruments. The S1, S2, F1, F2 and F3 ProTaper Universal files and the size 15, 0.06 taper, size 20, 0.06 taper, size 25, 0.06 taper, and size 30, 0.06 taper ProFile Vortex files produced an oval foramen in 71%, 71%, 85%, 85%, 71%, 71%, 85%, 85% and 89% of the cases, respectively.

Conclusions  In most samples, the ProTaper Universal and ProFile Vortex files produced transportation of the major foramen and created an oval-shaped major foramen after overinstrumentation.

Aim  To compare in vivo three different electronic root canal length measurement devices: Dentaport ZX, Raypex 5 and ProPex II.

Methodology  Thirty single-rooted permanent teeth scheduled for extraction because of periodontal disease were selected from 10 adult patients (ranging from 45 to 67 years) and divided into three groups of 10 teeth. Before the extraction, an access cavity was prepared and the crown was adjusted to establish a stable reference point for all measurements. The working length in Group 1 was determined using the Dentaport ZX apex locator. A K-file with the largest diameter that could reach the last green bar on the screen was stabilized in the canal using a dual-curable flow resin composite. The same procedure was used for the Raypex 5 (the file reached the last yellow bar) and Propex II (0.0 orange bar) apex locators. The teeth were then extracted and cleared. The distance between the tip of the file and the major foramen was then calculated for each tooth using digital photography according to Axiovision AC software (Carl Zeiss). Positive values were assigned when the file tip passed beyond the major foramen, negative values when the tip was short of the foramen and zero value when the file tip and the foramen coincided. Statistical analysis was performed using the chi-squared test or Fisher’s exact test (P ≤ 0.05).

Results  Dentaport ZX, Raypex 5 and ProPex ΙΙ produced, respectively, 6, 2 and 4 out of 10 correct measurements, 0, 6 and 5 long measurements and 4, 2, and 1 short measurements. The differences between the three electronic root canal length measurement devices were not significant (P = 0.507).

Conclusions  Under the in vivo conditions of this study, the three electronic root canal length measurement devices were not significantly different in terms of locating the major foramen.

Aim  To evaluate the microbial ecology of the coronal and apical segments of infected root canal systems using a complete sampling technique and next-generation sequencing.

Methodology  The roots of 23 extracted teeth with apical periodontitis were sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5-V6 region.

Results  The sequences were classified into 606 taxa (species or higher taxon), representing 24 bacterial phyla or candidate divisions and one archaeal phylum. Proteobacteria were more abundant in the apical samples (P < 0.05), whilst Actinobacteria were in significantly higher proportions in the coronal samples. The apical samples harboured statistically significantly more taxa than the coronal samples (P = 0.01) and showed a higher microbial diversity. Several taxa belonging to fastidious obligate anaerobes were significantly more abundant in the apical segments of the roots compared with their coronal counterparts.

Conclusions  Endodontic infections are more complex than reported previously. The apical part of the root canal system drives the selection of a more diverse and more anaerobic community than the coronal part. The presence of a distinct ecological niche in the apical region explains the difficulty of eradication of the infection and emphasizes the need for new treatment approaches to be developed.

Aim  To evaluate the influence of propylene glycol (PG) on the flowability, setting time, pH and calcium ion release of mineral trioxide aggregate (MTA).

Methodology  Mineral trioxide aggregate was mixed with different proportions of PG, as follows: group 1: MTA + 100% distilled water (DW); group 2: MTA + 80% DW and 20% PG; group 3: MTA + 50% DW and 50% PG; group 4: MTA + 20% DW and 80% PG; group 5: MTA + 100% PG. The ANSI/ADA No. 57 was followed for evaluating the flowability and the setting time was measured by using ASTM C266-08. For pH and calcium release analyses, 50 acrylic teeth with root-end cavities were filled with the materials (n = 10) and individually immersed in flasks containing 10 mL deionized water. After 3 h, 24 h, 72 h and 168 h, teeth were placed in new flasks and the water in which each specimen was immersed had its pH determined by a pH metre and the calcium release measured by an atomic absorption spectrophotometer with a calcium-specific hollow cathode lamp. Data were analysed by using one-way anova test for global comparison and by using Tukey’s test for individual comparisons.

Results  The highest value of flowability was observed with MTA + 20% DW and 80% PG and the lowest values were found with MTA + 100% DW. They were significantly different compared to the other groups (P < 0.05). The presence of PG did not affect the pH and calcium release. The MTA + 100% PG favoured the highest (P < 0.05) pH and calcium release after 3 h. Increasing the PG proportion interfered (P < 0.05) with the setting time; when used at the volume of 100% setting did not occur.

Conclusion  The addition of PG to MTA-Angelus increased its setting time, improved flowability and increased the pH and calcium ion release during the initial post-mixing periods. The ratio of 80% DW – 20% PG is recommended.

Aim  To compare the centring ability and the shaping ability of ProTaper (PT) files used in reciprocating motion and PT and Twisted Files (TF) used in continuous rotary motion, and to compare the volume changes obtained with the different instrumentation techniques using micro-computed tomography.

Methodology  Sixty mesial canals of thirty mandibular molars were randomly assigned to three instrumentation techniques: group 1, canals prepared with the PT series (up to F2) (n = 20); group 2, canals prepared with the F2 PT in reciprocating motion (n = 20); group 3 canals prepared with the TF series (size 25) (n = 20). Teeth were scanned pre- and post-operatively using micro-computed tomography to measure volume and shaping changes, and the obtained results were statistically analysed using parametric tests.

Results  The increase in canal volume obtained with the three instrumentation techniques was not significantly different. Canals were transported mostly towards the mesial aspect in the apical- and mid-third of the roots, and towards the furcal aspect coronally. No difference in the transportation and centring ratio was found between the techniques. There was no significant difference between the times of instrumentation (TF: 62.5 ± 5.4 s; PT: 60.6 ± 3.9 s; and F2 PT file in reciprocating motion: 51.0 ± 3.3 s).

Conclusions  ProTaper files used in reciprocating motion and PT and TF used in continuous rotary motion were capable of producing centred preparations with no substantial procedural errors.

Aim  To evaluate the fracture resistance and fracture patterns of root filled maxillary first premolar teeth (with mesio–occlusal cavity preparation) restored with several composite restoration designs.

Methodology  One hundred extracted sound human maxillary first premolars were randomly divided into five groups. Group 1 remained untreated (negative control). Conventional root canal treatment with additional mesial–occlusal cavity preparation was carried out on teeth in groups 2–5. In group 2, the teeth were restored intra-coronally with direct composite resin (positive control). In group 3, the palatal cusps of the teeth were reduced, and the cavities were restored with composite resin covering the palatal cusp (partial coverage). In group 4, the buccal and palatal cusps along with the distal marginal ridges were reduced; the cavities and cusps were restored with composite resin (conventional full coverage). In group 5, the buccal and palatal cusps were reduced but the distal marginal ridges were conserved. The cavities and the cusps were restored with composite resin (modified full coverage). All teeth were subjected to a progressive compressive loading parallel to their longitudinal axis until fracture. Fracture resistance was analysed using the one-way ANOVA and Fisher’s LSD test. Fracture patterns were analyzed with chi-square test. The significance level was set at 0.05.

Results  The fracture resistance (mean ± SD) of groups 1–5 was 1131 ± 207N, 904 ± 184N, 927 ± 224N, 1095 ± 289N and 1085 ± 243N, respectively (groups 1, 4, 5 > groups 2, 3; P = 0.004). Cusp fractures were recorded as the fracture pattern in 20 (100%), 19 (95%), 16 (80%), 8 (40%) and 12 (60%) premolars in groups 1–5, respectively (groups 1, 2 > groups 4, 5; group 3 > group 4; P < 0.001).

Conclusions  When direct composite resin was used to restore root filled maxillary first premolar teeth involving a proximal surface, those restored with full-coverage designs had greater fracture resistance.

Aim  To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7.

Methodology  Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferroni’s t-test for multi-group comparisons test.

Results  BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P < 0.05). BisGMA was found to induce two modes of cell death. The mode of cell death changed from apoptosis to necrosis as the concentrations of BisGMA elevated. Caspase-3, caspase-8 and caspase-9 activities were significantly induced by BisGMA in a dose-dependent manner (P < 0.05). Moreover, BisGMA exhibited genotoxicity via a dose-related increase in the numbers of micronucleus and DNA strand breaks (P < 0.05).

Conclusions  Cytotoxicity and genotoxicity induced by BisGMA are mediated by DNA damage and caspase activation.

Aim  To compare the antimicrobial efficacy of two-high power lasers (Nd:YAG and Er:YAG) and two commercial antimicrobial photodynamic therapy (aPDT) systems with that of sodium hypochlorite (NaOCl) action on Enterococcus faecalis biofilms grown on dentine discs.

Methodology Enterococcus faecalis biofilms were grown on dentine discs in a microtiter plate, incubated for 24 h and subjected to the following treatments: aPDT (Denfotex and Helbo system), Er:YAG laser irradiation (2940 nm, 50 mJ or 100 mJ, 15 Hz, 40 s), Nd:YAG laser irradiation (1064 nm, 2 W, 15 Hz, 40 s) and immersion in 2.5% (w/v) NaOCl for 1, 5, 10 and 30 min. Surviving bacteria were harvested, and the number of CFU per disc was determined by plate counting.

Results  Significant reductions (anova, P ≤ 0.05) in viable counts were observed for aPDT (Helbo) (2 log₁₀ reduction), Er:YAG irradiation using 100 mJ pulses (4.3 log₁₀ reduction) and all NaOCl treatments (>6 log₁₀ reduction). NaOCl (2.5%) for 5 min effectively eliminated all bacteria. aPDT (Denfotex), Er:YAG irradiation using 50 mJ pulses and Nd:YAG treatment caused a reduction in the viable counts of <1 log₁₀ unit; these results were not significantly different from the untreated controls.

Conclusion  Within the limitations of this particular laboratory set-up, NaOCl was the most effective in E. faecalis biofilm elimination, while Er:YAG laser treatment (100 mJ pulses) also resulted in high reductions in viable counts. The use of both commercial aPDT systems resulted in a weak reduction in the number of E. faecalis cells. Nd:YAG irradiation was the least effective.

Aim  To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model.

Methodology  Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor β (TGF-β) was assessed using real-time PCR. Levene’s test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05).

Results  The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-β mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-β mRNA expression during the chronic phase.

Conclusion  Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-β and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response.

Aim  To investigate the effect of cyclic fatigue on nickel-titanium (NiTi) endodontic instruments using a nano-indentation test.

Methodology  Eight ProFile NiTi rotary instruments (size 30, taper 0.06; Dentsply Maillefer, Ballaigues, Switzerland) were tested using a cyclic fatigue set-up until fracture. The fractured instruments and eight new NiTi instruments of the same size and taper were used for a nano-indentation test on the internal surfaces of a NiTi instruments in the region just adjacent to their fractured edge (group I) and in the same region of the new group (group II), and the cutting part beside the shaft for both instruments [group III (fractured) and group IV (new)]. Data were statistically analyzed using one-way analysis of variance and Games–Howell post hoc test. The alpha-type error was set at 0.05.

Results  Significant differences in terms of hardness and elastic modulus for each group (P < 0.05) were found, with group I having the lowest mean values followed by group III. Additionally, standard deviations increased remarkably after failure, as represented by groups I and III.

Conclusion  The nano-indentation technique can be applied to determine the performance and the failure mechanism of NiTi instruments. The fatigue process revealed a significant decrease in the hardness and elastic modulus of the NiTi instrument. As indicated by the low hardness, the fatigue process did not result in work hardening but rather work softening.

Aim  To assess the effectiveness of three systems of mechanical preparation to reduce Enterococcus faecalis within root canals.

Methodology  Twenty-four human single-rooted canine teeth were standardized to a length of 17 mm and the canal contents removed using a size 20 K-file, as the last apical file. After irrigation and sterilization, the canals were contaminated with E. faecalis and incubated for 21 days at 37 °C with 5% CO₂. Then, the teeth were divided into three groups for mechanical preparation with: ProTaper rotary system, ProTaper manual system and manual K-files. Samples of the root canal contents, before and after the debridement, were collected with sterile paper points for 1 min. Then, the samples were diluted and plated in Brain Heart Infusion (BHI) agar. The colony-forming units were counted and the percentage reduction calculated. The reduction and log CFU mL⁻¹ were compared between groups using Wilcoxon nonparametric test and two-way analysis of variance, respectively.

Results  There was a significant reduction in the number of CFU/mL (P = 0.000) before and after debridement for all the systems used. However, there was no significant difference between the systems.

Conclusion  All the three instrumentation systems reduced E. faecalis counts to a similar degree.

Aim  To compare physical and mechanical properties of one conventional and one thermomechanically treated nickel–titanium (NiTi) wire used to manufacture rotary endodontic instruments.

Methodology  Two NiTi wires 1.0 mm in diameter were characterized; one of them, C-wire (CW), was processed in the conventional manner, and the other, termed M-Wire (MW), received an additional heat treatment according to the manufacturer. Chemical composition was determined by energy-dispersive X-ray spectroscopy, phase constitution by XRD and the transformation temperatures by DSC. Tensile loading/unloading tests and Vickers microhardness measurements were performed to assess the mechanical behaviour. Data were analysed using analysis of variance (α = 0.05).

Results  The two wires showed approximately the same chemical composition, close to the 1 : 1 atomic ratio, and the β-phase was the predominant phase present. B19′ martensite and the R-phase were found in MW, in agreement with the higher transformation temperatures found in this wire compared with CW, whose transformation temperatures were below room temperature. Average Vickers microhardness values were similar for MW and CW (P = 0.91). The stress at the transformation plateau in the tensile load–unload curves was lower and more uniform in the M-Wire, which also showed the smallest stress hysteresis and apparent elastic modulus.

Conclusions  The M-Wire had physical and mechanical properties that can render endodontic instruments more flexible and fatigue resistant than those made with conventionally processed NiTi wires.

Aim  To compare shaping ability and cleaning effectiveness of two reciprocating single-file systems with Mtwo and ProTaper rotary instruments during the preparation of curved root canals in extracted teeth.

Methodology  A total of 80 root canals with curvatures ranging between 25° and 39° were divided into four groups of 20 canals. Based on radiographs taken prior to instrumentation, the groups were balanced with respect to the angle and the radius of canal curvature. Canals were prepared to the following apical sizes: Mtwo: size 35 using the single-length technique; ProTaper: F3, instruments were used in a modified crown-down manner; Reciproc and WaveOne: size 25. Using pre- and post-instrumentation radiographs, straightening of the canal curvatures was determined with a computer image analysis program. Preparation time and instrument failures were also recorded. These data were analysed statistically using anova and Student–Newman–Keuls test. The amounts of debris and smear layer were quantified on the basis of a numerical evaluation scale and were analysed statistically using the Kruskal–Wallis test.

Results  During preparation no file fractured. All instruments maintained the original canal curvature well with no significant differences between the different files (P = 0.382). Instrumentation with Reciproc was significantly faster than with all other instruments (P < 0.05), while WaveOne was significantly faster than Mtwo and ProTaper (P < 0.05). For debris removal, Mtwo and Reciproc instruments achieved significantly better results (P < 0.05) than the other instruments in the apical third of the canals. In the middle and coronal parts, no significant differences were obtained between Mtwo, Reciproc and WaveOne (P > 0.05), while ProTaper showed significantly more residual debris (P < 0.05). The results for remaining smear layer were similar and not significantly different for the different parts of the canals (P > 0.05).

Conclusions  Under the conditions of this study, all instruments maintained the original canal curvature well and were safe to use. The use of Mtwo and Reciproc instruments resulted in better canal cleanliness in the apical part compared with ProTaper and WaveOne.

Aim  To assess the ability of a recently developed tricalcium silicate-based cement (Biodentine™) to induce reparative dentine synthesis and to investigate its capacity to modulate pulp cells TGF-β1 secretion.

Methodology  Biodentine™ was directly applied onto the dental pulp in an entire human tooth culture model. After various culture periods, the interaction of the material with dental pulp tissue was analysed on tissue sections. The effect of increasing surface area of this material on TGF-β1 secretion was investigated on pulp cell cultures and compared with that of MTA, calcium hydroxide and Xeno^®III adhesive resin. After performing artificial injuries on pulp cell cultures, the materials eluates were added for 24 h and then TGF-β1 secretion was quantified by ELISA. Controls were performed by incubating intact cells with the culture medium, while injured cells TGF-β1 level was used as the baseline value.

Results  Biodentine™ induced mineralized foci formation early after its application. The mineralization appeared under the form of osteodentine and expressed markers of odontoblasts. Biodentine™ significantly increased TGF-β1 secretion from pulp cells (P < 0.03) independently of the contact surface increase. This increase was also observed with calcium hydroxide and MTA, but not with the resinous Xeno^®III. The statistical analysis showed statistically significant differences between capping materials and the resinous Xeno^®III (P < 0.001).

Conclusions  When Biodentine™ was applied directly onto the pulp, it induced an early form of reparative dentine synthesis, probably due to a modulation of pulp cell TGF-β1 secretion.

Aim  To evaluate the force required to fracture roots vertically after the removal of broken instruments using ultrasonic tips and a Masserann kit.

Methodology  Thirty-nine extracted human anterior teeth with single and straight roots were used. The crown of each tooth was removed until the full length of the tooth was 13 mm. All canals were instrumented with the step-back technique to a size 25 K-file. The teeth were randomly divided into three groups: two experimental groups and a control group. The size 30 K-files used for the test were notched to a depth of half of the instrument with a No. 2 round bur at a point 3 mm from the tip to facilitate breakage of the file at this point. The fractured instruments were removed from the canals using ultrasonic tips or a Masserann kit. The samples were subjected to a continual vertical loading, using a universal testing machine. For each root, the force at the time of fracture was recorded in Newtons. The results were analysed statistically using one-way anova and Tukey HSD test.

Results  The force required to fracture the roots vertically was significantly higher in the control group than the experimental groups (P < 0.01). The roots from which the broken instruments had been removed using ultrasonic tips required more force to fracture than roots in the Masserann group; however, the difference was not statistically significant.

Conclusion  Removal of a fractured instrument from the middle third of the root decreased the force required to fracture the root vertically, regardless of the technique used for instrument removal. There was not difference between the ultrasonic technique and the Masserann technique.

Aim  To evaluate the accumulation of hard-tissue debris when using the Self-adjusting File (SAF) system in mesial roots of mandibular molars with isthmuses and to compare it with that occurring when the ProTaper file system was used.

Methodology  Forty extracted human mandibular molars with joining mesial root canals and an isthmus between the two canals were randomly assigned to two experimental groups and scanned using micro-computed tomography. Root canals in the control group (N = 20) were instrumented using the ProTaper rotary system until F3. Irrigation with 1 mL of 3% NaOCl solution was applied after each instrument. Root canals in the experimental group (N = 20) were prepared using the SAF for 4 min, with continuous irrigation (3% NaOCl, 4 mL min⁻¹). After rescanning, canals in both groups were further irrigated with 5 mL of 17% EDTA for 2 min. After final scanning, the per cent value of total canal system volume filled with hard-tissue debris was calculated. Statistical analysis was performed using Mann–Whitney U-test; the alpha-type error was set at 1%.

Results  Instrumentation of the root canals with ProTaper left 10.1% (IQR 5.2) of the total canal system volume filled with hard-tissue debris while preparation with the SAF left 1.7% (IQR 1.6). This difference was highly significant (P < 0.0001). After subsequent EDTA irrigation, these values were significantly reduced (P < 0.01) to 7.9% (IQR 4.1) and 1.3% (IQR 0.8) in the ProTaper and SAF groups, respectively.

Conclusion  Preparation with the SAF system resulted in less hard-tissue debris accumulation in isthmus-containing root canal systems compared with instrumentation with ProTaper rotary files.

Aim  To compare the cleaning effectiveness of manual, hybrid and rotary instrumentation techniques in primary molar teeth.

Methodology  Fifteen primary molars were selected. After endodontic access, the teeth were immersed in a medium containing Enterococcus faecalis and divided into three groups, according to the root canal instrumentation technique: group 1 – manual, group 2 – hybrid and group 3 – nickel–titanium (NiTi) rotary files. For microbiological evaluation, comparisons before and after instrumentation were performed using the paired Student’s t-test. One-way anova complemented with the Student’s t-test was used to compare the percentage of microbial reduction. Instrumentation time was evaluated by Kruskal–Wallis and Student–Newman–Keuls tests. Images obtained under scanning electron microscopy were analysed by three blinded examiners, and kappa statistics was used to evaluate calibration among examiners. The most frequent results among examiners were analysed using Kruskal–Wallis and Student–Newman–Keuls tests.

Results  The hybrid technique required a significantly longer instrumentation time than the manual and rotary techniques (P < 0.05). All techniques tested were able to significantly reduce the number of E. faecalis (P < 0.05). The hybrid technique was associated with the highest intracanal bacterial reduction, with a statistically significant difference compared with manual instrumentation (P = 0.01). Manual instrumentation resulted in the lowest amount of debris and the highest amount of smear layer when compared with the rotary and hybrid techniques (P < 0.05). There was no difference between rotary and hybrid instrumentation in the removal of debris and smear layer.

Conclusion  The use of NiTi rotary files is an option for root canal instrumentation in primary teeth.

Aim  To compare the prevalence of periapical lesions on individual roots viewed with intraoral (periapical) radiographs and cone-beam computed tomography (CBCT) of teeth treatment planned for endodontic treatment.

Methodology  Diagnostic intraoral and CBCT scans were taken of 151 teeth in 132 patients diagnosed with primary endodontic disease. The presence or absence of periapical lesions was assessed by a consensus panel consisting of two calibrated examiners, a consensus agreement was reached if there was any disagreement. The panel viewed the images under standardised conditions.

Results  Two hundred and seventy-three paired roots were assessed with both radiological systems, periapical lesions were present in 55 (20%) and absent in 218 (80%) roots assessed with periapical radiographs. When the same 273 sets of roots were assessed with CBCT, lesions were present in 130 (48%) and absent in 143 (52%) roots. Seventy-five additional roots were detected with CBCT.

Conclusion  The limitations of intraoral radiographs which may hinder the detection of periapical lesions are overcome with CBCT. This results in firstly, more roots being assessed, and secondly, more periapical lesions being detected with CBCT.

Aim  To compare the changes in the surface structure and elemental distribution, as well as the percentage of ion release, of four calcium silicate-containing endodontic materials with a well-established epoxy resin-based sealer, submitted to a solubility test.

Methodology  Solubility of AH Plus, iRoot SP, MTA Fillapex, Sealapex and MTA-Angelus (MTA-A) was tested according to ANSI/ADA Specification 57. The deionized water used in the solubility test was submitted to atomic absorption spectrophotometry to determine and quantify Ca²⁺, Na⁺, K⁺, Zn²⁺, Ni²⁺ and Pb²⁺ ions release. In addition, the outer and inner surfaces of nonsubmitted and submitted samples of each material to the solubility test were analysed by means of scanning electron microscopy and energy-dispersive spectroscopy (SEM/EDX). Statistical analysis was performed by using one-way anova and Tukey’s post hoc tests (α = 0.05).

Results  Solubility results, in percentage, sorted in an increasing order were −1.24 ± 0.19 (MTA-A), 0.28 ± 0.08 (AH Plus), 5.65 ± 0.80 (Sealapex), 14.89 ± 0.73 (MTA Fillapex) and 20.64 ± 1.42 (iRoot SP). AH Plus and MTA-A were statistically similar (P > 0.05), but different from the other materials (P < 0.05). High levels of Ca²⁺ ion release were observed in all groups except AH Plus sealer. MTA-A also had the highest release of Na²⁺ and K⁺ ions. Zn⁺² ion release was observed only with AH Plus and Sealapex sealers. After the solubility test, all surfaces had morphological changes. The loss of matrix was evident and the filler particles were more distinguishable. EDX analysis displayed high levels of calcium and carbon at the surface of Sealapex, MTA Fillapex and iRoot SP.

Conclusions  AH Plus and MTA-A were in accordance with ANSI/ADA’s requirements regarding solubility whilst iRoot SP, MTA Fillapex and Sealapex did not fulfil ANSI/ADA’s protocols. High levels of Ca²⁺ ion release were observed in all materials except AH Plus. SEM/EDX analysis revealed that all samples had morphological changes in both outer and inner surfaces after the solubility test. High levels of calcium and carbon were also observed at the surface of all materials except AH Plus and MTA-A.

Aim  To determine the response of dental pulp stem cells (DPSCs) to DNA-damaging cytostatic cisplatin and compare it with the response of normal human dermal fibroblasts (HDFs).

Methodology  Dental pulp stem cells were exposed to 5, 10, 20 or 40 μmol L⁻¹ of cisplatin. The proliferation of affected cells was assessed by a Z2 Counter and viability was assessed by means of a Vi-Cell XR using Trypan blue exclusion staining. Cell cycle analysis and induction of apoptosis were performed by flow cytometry. Induction of apoptosis was determined by monitoring the activities of caspases. The expression of proteins was detected by electrophoresis and Western blotting. The descriptive statistics of the results was analyzed by Student’s t-test.

Results  Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. All three main Mitogen-activated protein kinases (MAPK) families – extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinase (JNK) and p38 were activated after treatment of DPSCs with cisplatin. The activation of MAPK pathways was not observed in HDFs exposed to cisplatin. The exposure of DPSCs and HDFs to cisplatin provoked an increase in p53 and p21 expression and p53 phosphorylation of serine 15. Higher concentrations of cisplatin reduced the viability of DPSCs and HDFs and induced the activation of caspases 3/7 and 9.

Conclusion  Dental pulp stem cells had a greater genotoxic stress response to cisplatin compared to HDFs. Cisplatin in higher concentrations triggered activation of MAPK and apoptosis in DPSCs but not in HDFs.

Aim  To investigate alterations in Na_V1.8 and Na_V1.9 expression within inflamed dental pulp tissue of human primary teeth.

Methodology  Dental pulp tissue obtained from both normal and inflamed pulps in primary teeth as well as pulps from normal and inflamed permanent teeth was used. The quantity of Na_V1.8 and Na_V1.9 expression in the dental pulp tissue was investigated using Western blot analysis. General neuron marker (PGP9.5) was used to quantify for neural density, and an increase in metalloproteinase-9 was used to indicate pulpal inflammation in inflamed teeth. Statistically significant differences for each determined parameter between normal and inflamed teeth of both primary and permanent teeth were tested using the Mann–Whitney rank sum test.

Results  There was no significant difference in neural density of normal and inflamed dental pulp tissue, although degrees of inflammation were increased in the inflamed dental pulp of both permanent and primary teeth (P < 0.05). Na_V1.8 and Na_V1.9 expression in inflamed pulps of permanent teeth increased significantly compared with normal permanent teeth (P < 0.05). However, only Na_V1.8 expression was increased significantly in the inflamed dental pulp of primary teeth (P < 0.05).

Conclusions  Na_V1.8 alone may be the therapeutic target for treatment of painful pulpitis in primary teeth.

Aim  To assess in a laboratory experimental model the efficacy of a novel root canal irrigant, QMiX, against Enterococcus faecalis and mixed plaque bacteria in planktonic phase and biofilms. In addition, its ability to remove smear layer was examined.

Methodology Enterococcus faecalis and mixed plaque bacteria were exposed to QMiX, 2% chlorhexidine (CHX), MTAD and 1% sodium hypochlorite (NaOCl) for 5 s, 30 s and 3 min. Following exposure, samples were taken, serially diluted and grown aerobically and anaerobically on tryptic soy agar (TSA) plates or on blood agar plates for 24 and 72 h, respectively, to measure killing of bacteria. E. faecalis and plaque biofilms were grown for 3 weeks on collagen-coated hydroxyapatite or dentine discs and exposed for 1 and 3 min to QMiX, 2% CHX, MTAD, 1% and 2% NaOCl. The amount of killed bacteria in biofilms was analysed by confocal laser scanning microscopy using viability staining. Dentine blocks were exposed to QMiX and 17% EDTA for 5 min. The effectiveness of smear layer removal by the solution was evaluated using scanning electron microscopy. For statistical analysis, one-way analysis of variance and comparison of two proportions were used.

Results  QMiX and 1% NaOCl killed all planktonic E. faecalis and plaque bacteria in 5 s, while 2% CHX and MTAD were unable to kill all plaque bacteria in 30 s, and some E. faecalis cells survived even 3 min of exposure. QMiX and 2% NaOCl killed up to 12 times more biofilm bacteria than 1% NaOCl (P < 0.01), 2% CHX (P < 0.05; P < 0.001) and MTAD (P < 0.05; P < 0.001). QMiX removed smear layer equally well as EDTA (P = 0.18 × 10⁻⁵).

Conclusion  QMiX and NaOCl were superior to CHX and MTAD under laboratory conditions in killing E. faecalis and plaque bacteria in planktonic and biofilm culture. Ability to remove smear layer by QMiX was comparable to EDTA.

Aim  To evaluate the antimicrobial efficacy of calcium hydroxide-based intracanal medicaments against Enterococcus faecalis.

Methodology  One hundred and six single-rooted human teeth were inoculated with E. faecalis (ATCC 29212) and incubated in an microaerophilic environment for 21 days. Teeth were divided according to the medicaments and period: G1 – Calen (7 days); G2 – Calen (14 days); G3 – Calen/camphorated paramonochlorophenol (CMCP) (7 days); G4 – Calen/CMCP (14 days); G5 – Calen/0.4% chlorhexidine (CHX) (7 days); G6 – Calen/0.4% CHX (14 days); G7 – Calen/1% CHX (7 days); G8 – Calen/1% CHX (14 days); G9 – chemo-mechanical preparation with no medication and G10 – no chemo-mechanical preparation and no medication. Microbial samples were harvested immediately after removal of the intracanal dressing and 7 days later. Data were subjected to anova (P < 0.05).

Results  All medicaments were associated with a reduction in E. faecalis after removal of the dressing (post-medication harvest). However, all specimens were associated with an increase in CFU mL⁻¹ values after 7 days (final harvest). Calen/CMCP and Calen/CHX had the lowest CFU mL⁻¹ values (P < 0.05).

Conclusion  In this laboratory study using extracted teeth all calcium hydroxide-based medicaments were able to significantly reduce the CFU mL⁻¹ values of E. faecalis in the root canal system.

Aim  To evaluate the effect of intracanal dentine wettability on human dental pulp cell (HDPC) attachment after application of endodontic irrigants.

Methodology  Twenty extracted human premolar teeth with straight canals were sectioned at the apical and coronal thirds of each root, and the remaining mid-root portions were bisected longitudinally. After polishing, the specimens were divided into two groups for wettability and cell attachment measurements, respectively. The intracanal surface wettability was assessed by atomic force microscopy after a rinse of H₂O (control), 5.25% sodium hypochlorite (NaOCl), 17% ethylenediaminetetraacetic acid (EDTA) and MTAD (n = 5). Additionally, HDPCs were seeded onto the irrigated root canal dentine and the cell attachment was evaluated by calcein-CAM and propidium iodide (PI) dying under fluorescence microscope (n = 5). Data were analysed by means of one-way anova and LSD test.

Results  Compared with the control group, 5.25% NaOCl, 17% EDTA and MTAD treatment significantly increased surface wettability (P < 0.001). The rank order of adhesion force was MTAD >17% EDTA >5.25% NaOCl > H₂O. The highest average numbers of viable HDPCs were observed on the root canals irrigated with MTAD, followed by the 17% EDTA group (P < 0.001), and the lowest average numbers of viable cells were detected in the 5.25% NaOCl and H₂O groups.

Conclusions  Surface wettability was affected by the irrigants. Increasing in wettability produced higher levels of HDPC attachment to irrigated dentine.

Aim  To describe the successful non-surgical endodontic management of a permanent maxillary lateral incisor tooth with four root canals.

Summary  Aberrant morphology of root canals can occur in any tooth, including maxillary lateral incisors. In the presented case, clinical examination and radiographs clearly depicted the presence of four root canals in a right maxillary lateral incisor. The differential diagnosis included fusion, gemination, dens invaginatus or a combination of these. The report emphasises the need for attention during root canal treatment of maxillary lateral incisors.

Key learning points 

• •
   Clinicians should be aware of the variable root canal anatomy in maxillary lateral incisor teeth.
• •
   Careful examination of radiographs and the internal anatomy of teeth are essential prerequisites for successful root canal treatment.
• •
   Four root canals in maxillary lateral incisors is a possibility.

Aim  To examine the effects of altering the Si/Ca molar ratio (6 : 4, 5 : 5, and 4 : 6) of a quick-setting calcium silicate cement on in vitro cell attachment.

Methodology  Working time and setting time of three different calcium silicate cements were measured. Alamar Blue was used for real-time and repeated monitoring of cell attachment and proliferation. The Si and Ca ion concentrations of the cell culture medium in the presence of three different calcium silicate cements seeded with MG63 cells were measured. Kinetic immunofluorescent staining of MG63 cells was performed during cell attachment and spreading. Reverse transcription-polymerase chain reaction was employed to determine gene expression in MG63 cells cultured on the cements. One-way analysis of variance was used to evaluate the significance of the differences between the mean values.

Results  The working time (4–7 min) and setting time (17–24 min) of the cements were shortened with an increase in the Ca content of the calcium silicate powders after mixing the powder with water. In contrast, the higher the Si content in the cement, the more the MG63 cells attached to the cement at all culture time-points, accompanying by the formation of more obvious actin stress fibres. Cell proliferation and differentiation increased significantly (P < 0.05) with an increase in the Si content of the calcium silicate cements. Si ion concentration of the culture medium increased significantly (P < 0.05) with increasing cement Si content and culture time-points.

Conclusions  The higher Si content cement enhanced the higher expression of cell attachment, proliferation and differentiation as compared to the lower Si content cement.

Aim  To investigate the physical properties of tricalcium silicate (TCS) with and without the addition of a radiopacifier and compare them with that of Portland cement (PC) and radiopaque PC in an mineral trioxide aggregate-like system.

Methodology  Tricalcium silicate, PC and radiopacified variants containing 20% bismuth oxide were tested for radiopacity, compressive strength, setting time and dimensional stability. All the testing was performed at 37 °C and under different environmental conditions namely at 100% humidity or immersed in either water or Hank’s balanced salt solution (HBSS). Testing was performed after both 1 and 28 days.

Results  The cements exhibited radiopacity values equivalent to <3 mm. Addition of 20% bismuth oxide resulted in adequate radiopacity. The strength of TCS was independent of the curing conditions. The cements without radiopacifier had improved strength characteristics when immersed in HBSS, whilst the radiopacified cements exhibited higher strengths when soaked in water. Tricalcium silicate demonstrated the shortest setting time. Addition of bismuth oxide increased the setting time of the cements while HBSS inhibited the setting of bismuth oxide-replaced cements. The PC-based materials exhibited a net contraction higher than that recorded for TCS-based cements in all curing conditions. The dimensional change exhibited by the specimens was generally greater in the first few hours of setting, but then stabilized with time.

Conclusions  Tricalcium silicate cement required the addition of a radiopacifying agent to make it suitable for use as a dental material. Tricalcium silicate exhibited adequate physical properties and thus was shown to be a suitable replacement for the PC component in MTA. Bismuth oxide drastically increased the setting time of the test cements in phosphate-containing solutions. Alternative radiopacifiers that do not retard the setting time need to be investigated.

Aim  To develop a simple, practical and inexpensive model, which enables the use of electronic apex locators (EALs) during pre-clinical and continuing education.

Methodology  Extracted teeth were placed in a mould and embedded in acrylic resin. The resin was applied in two consecutive steps to form a cavity around the root apices. A closable plastic tube serves as a valve, and a steel wire connects to the EAL.

Results  With its semi-closed reservoir for conductive fluids surrounding the root apices, the new model enables working length measurements of root canals using EALs.

Conclusions  The model simulates the clinical situation for endodontic teaching purposes, as it allows working length determination of root canals as recommended. The measuring results of the EAL can be verified by radiography. At the same time, the roots are not directly visible and accessible to the user, allowing a precise evaluation and grading of the treatment.

Aim  To collect quantitative information about the numbers of odontoblast-like cells and reparative dentine thickness after direct pulp capping with platelet-rich plasma (PRP) and enamel matrix derivative (EMD).

Methodology  The experiment was conducted on 36 Wistar albino rats and a total of 144 incisor teeth. Calcium hydroxide, mineral trioxide aggregate, PRP and EMD were applied as direct capping agents on the pulps of 96 incisors (n = 24). Positive and negative control groups were created on the remaining 48 incisors. The teeth were extracted on the 7th and 28th days. After routine histological preparation, cross-sections were stained with haematoxylin and eosin. The numbers of the odontoblast-like cells were measured histomorphometrically on day 7 and day 28. The thickness of the reparative dentine was also measured. The number of odontoblast-like cells was also measured beneath the dentine bridge. The normal distribution of all data was tested with the Mann–Whitney U test. The statistical differences between groups were analysed using the Kruskal–Wallis test.

Results  The mean number of odontoblast-like cells increased between day 7 and 28 following pulp exposure (P > 0.01) in all groups except for the EMD group (P < 0.01), when compared with both the experimental and negative control groups (P < 0.01). Reparative dentine formation was observed in all groups of teeth (P > 0.01).

Conclusions  Reparative dentine formation was observed, but with no significant difference between the groups. Odontoblast-like cells were observed in association with the outcome of pulps capped with PRP and EMD. PRP and EMD are possible capping agents that influence the thickness of reparative dentine formation.

Aim  To evaluate parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) expression in odontogenic cystic lesions and to compare immunoexpression between the lesions.

Methodology  Thirty-five radicular cysts, 22 dentigerous cysts and 17 keratocystic odontogenic tumours were evaluated. Immunohistochemical reactions against PHTR1 were carried out in 3-μm histological sections, and the expression and the intensity of PTHR1 expression were evaluated. For statistical analysis, the Fisher exact test was used, with a significance of 5%.

Results  The intensity of expression in the epithelial lining was significantly weaker in the radicular cyst (P = 0.007). However, in the fibrous capsule, the radicular cyst presented higher positivity for PTHR1 (P = 0.04).

Conclusions  The probable co-expression of PTHrP and PTHR1 in odontogenic cystic lesions may eventually have an autocrine and/or paracrine stimulus in the epithelial and mesenchymal cells, inducing proliferation and lesion growth.

Aim  To investigate the effect of orthognathic surgery, in particular genioplasty, on pulp sensibility and pulp blood flow (PBF).

Methodology  Pulp blood flow monitoring and CO₂ pulp sensibility testing were performed on 101 mandibular anterior teeth from 17 subjects who received mandibular bilateral sagittal split osteotomy (BSSO) with or without genioplasty and other maxillary surgical procedures combined with orthodontic treatment. A laser Doppler flowmeter was used to monitor PBF using customized acrylic splints for each subject. Preoperative baseline scores were recorded from up to 1 week preoperatively, and subsequently monitored for up to 18–28 weeks postoperatively. The data were analysed by RMANOVA and pairwise comparisons. Statistical significance was defined as P < 0.05.

Results  The average PBF in subjects who received genioplasty was not significantly different to those who received no genioplasty (F = 0.076, P = 0.784). In both groups, PBF decreased significantly postoperatively (F = 23.323, P = 0.000) and remained significantly lower (P = 0.000) than preoperative values at all times. PBF decreased markedly for 1–2 weeks postoperatively in patients who received genioplasty. However, there were no statistical differences between the PBF values at any time postoperatively for either group (P > 0.05). All teeth in both groups responded to CO₂ preoperatively. The average number of teeth that responded to CO₂ postoperatively in the genioplasty group was lower without significant difference when compared to the group without genioplasty (binomial distribution F = 2.63, P = 0.1256, normal distribution F = 2.98, P = 0.1048). At each progressive period after 1–2 weeks following surgery, the number of teeth responding to CO₂ increased significantly (P < 0.001) in both groups. At the end of the study, in the genioplasty group 81.1% of teeth responded to CO₂ compared to all teeth in the group without genioplasty.

Conclusion  Pulp blood flow decreased and remained significantly lower than preoperative values in mandibular anterior teeth postoperatively with or without genioplasty. At no time was the absence of PBF recorded. The number of teeth that responded to CO₂ decreased markedly postoperatively but recovered significantly over time. All teeth regained sensibility in the group without genioplasty at the end of the study. There were neither common trends nor coincidence in the recovery of PBF and pulp sensibility.

Aim  To investigate whether the p38α mitogen-activated protein kinases (MAPK) is involved in bone morphogenetic protein (BMP)-2-induced odontoblastic differentiation of human dental pulp cells (HDPCs).

Methodology  Recombinant retrovirus encoding shRNA against p38α MAPK was constructed to investigate the role of p38α MAPK on BMP-2-induced odontoblastic differentiation of HDPCs. HDPCs were transfected with retrovirus expressing sh-p38α. Activation of p38α MAPK was detected by Western blot. The effects of p38α MAPK on BMP-2-induced odontoblastic differentiation of HDPCs were measured by alkaline phosphatase (ALP) activity, and the expression of odontoblastic markers was identified by quantitative real-time polymerase chain reaction analysis. The effect of SD-282, a p38a-specific inhibitor, on BMP-2-induced odontoblastic differentiation was also investigated.

Results  BMP-2 dose- and time-dependently upregulated phosphorylation of p38α of HDPCs. Compared with BMP-2-treatment group, gene knock-down of p38α MAPK significantly inhibited ALP activity and the formation of mineralized nodules in HDPCs. Moreover, suppression of p38α MAPK repressed the odontoblastic differentiation in HDPCs. Consistently, inhibition of p38α by SD-282 also decreased odontoblastic differentiation.

Conclusions  p38α MAPK is involved in BMP-2-induced odontoblastic differentiation of HDPCs.

Aim  To evaluate the diagnosis of simulated external root resorption in cone beam computed tomography (CBCT) images acquired with different voxel sizes, then reconstructed with the same voxel size.

Methodology  Buccal, mesial and distal root surfaces of twenty single-rooted premolar teeth were evaluated for the presence or absence of simulated resorption defects. Images were obtained using a Classic i-CAT CBCT unit with two different voxel sizes (0.25 and 0.30 mm). Then all images were reconstructed to 0.25 mm voxel size. The McNemar’s test was applied to evaluate the agreement between the two protocols (different voxel sizes/reconstruction parameters) with the gold standard (defects created in the root). Accuracy, sensitivity, specificity, positive and negative predictive values were calculated for each voxel and defect sizes.

Results  Better sensitivity and accuracy were obtained with 0.25 (0.25 mm) voxel size. Specificity, positive and negative predictive values were highest with the 0.25 (0.30 mm) voxel size. McNemar’s test results revealed significant differences between the protocols with the gold standard (P < 0.0001). The results revealed that as the cavity size increased, sensitivity, accuracy, positive and negative predictive values also increased. When correlating the voxel size and root third, better sensitivity, accuracy, positive and negative predictive values were obtained in the middle third.

Conclusions  In the diagnosis of simulated external root resorption with the Classic i-CAT CBCT unit, images acquired with 0.30 mm voxel size and reconstructed with 0.25 mm voxel size provided the best protocol with a lower radiation dose than the 0.25 mm voxel size.

Aim  To compare the effect of several materials on the attachment of periodontal ligament (PDL) fibroblasts to experimentally perforated root surfaces.

Methodology  Root specimens (size 5 × 5 mm) were obtained from extracted human molar teeth and perforations with a 1 mm diameter were created. One group was kept as a control and the rest were repaired with the following materials: Amalgam, Dyract, IRM, Super Bond C&B and Mineral trioxide aggregate (MTA). PDL fibroblasts were placed at a density of 8 × 10⁴ cells on the root specimens, incubated on tissue culture inserts (48 h) and then transferred to 48 well-plates. MTT assays were performed at 48 and 96 h for PDL fibroblast survival. Cell attachment was observed using confocal microscopy on days 2 and 5. Total RNAs from the root specimens were isolated on day 5 and type I collagen (COL I) and Runt-related transcription factor 2 (Runx2) mRNA expressions were checked using Quantitative-Polymerase Chain Reaction (QPCR). For the MTT assay and QPCR, one-way analysis of variance (anova) and Tukey HSD multiple comparison tests were used to compare the groups.

Results  Mineral trioxide aggregate resulted in a significantly higher cell density (P < 0.001). Dyract, IRM and Super Bond C&B groups had a lower cell density when compared with the control and MTA groups at 48 h (P < 0.001). Confocal microscopy revealed that, among the experimental groups, the MTA group had the largest viable cell population over the restoration site when compared with the other materials; however, reduced cell attachment was noted in all groups when compared with the control. Increased Runx2 mRNA expressions were noted in MTA (P < 0.001) and IRM (P < 0.01) groups when compared with control and other tested materials. COL I transcripts were increased in IRM (P < 0.01), D, C&B and MTA (P < 0.001) when compared with the control.

Conclusion  Mineral trioxide aggregate provided a more favorable environment for PDL cell adhesion and growth.

Aim  To compare by means of a push-out test the interfacial strength of a dual-curing resin cement and a light-curing self-adhering resin composite when used in translucent fibre post cementation.

Methodology  Thirty-four extracted human premolars with single canals were selected and root filled. Translucent fibre posts (RelyX Fiber Post) were luted into the root canal using two resinous luting systems (n = 17). Dual-Curing Technique (DC): the specimens were treated with Excite DSC and RelyX ARC, which were light-cured simultaneously through the post for 60 s. Light-Curing Self-Adhering Technique (LCSA): the specimens were treated with Vertise Flow, which was light-cured through the post for 60 s. The specimens were sectioned transversally into six slices to perform the push-out test at the coronal, middle and apical regions of the root canals. Data were analysed by two-way anova. All specimens were analysed by stereomicroscopy and SEM to determine fracture patterns.

Results  There were no significant differences between the DC and LCSA techniques (P = 0.703) in any of the canal regions. Root region was not a significant factor for push-out values (P = 0.255) and group–region interactions were not significant (P = 0.740). For the DC technique, the majority of the fracture patterns (73.3%) were adhesive at the interface between dual-curing resin cement and adhesive. For the LCSA technique, the majority of the fracture patterns (71.7%) were adhesive at the interface between light-curing self-adhering resin composite and dentine.

Conclusions  The interfacial strength between light-curing self-adhering resin composite and root canal walls is equivalent to the interfacial strength between dual-curing cement and root canal walls.

Aim  To develop a new approach for locating the minor apical foramen (AF) using feature-extracting procedures from radiographs and then processing data using an artificial neural network (ANN) as a decision-making system.

Methodology  Fifty straight single-rooted teeth were selected and placed in a socket within the alveolar bone of a dried skull. Access cavities were prepared and a file was place in the canals to determine the working length. A radiograph was taken to evaluate the location of the file in relation to the minor foramen and further checked after retrieving the tooth from the alveolar socket. The location of the file tip was categorized into: beyond the AF (long), within the root canal (short) and just at the minor AF (exact). Each radiograph was used to extract relevant features using K-means, Otsu method and Wavelet protocol. Thirty-six extracted features were used for training and the rest were used for evaluating the multi-layer Perceptron ANN model.

Results  Analysis of the images from radiographs (test samples) by ANN showed that in 93% of the samples, the location of the AF had been determined correctly by false rejection and acceptation error methods.

Conclusion  Artificial neural networks can act as a second opinion to locate the AF on radiographs to enhance the accuracy of working length determination by radiography. In addition, ANN can function as a decision-making system in various similar clinical situations.

Aim  To evaluate the effect of four tooth storage temperature-based methods on quality of RNA obtained from cells retrieved from human dental pulps and human pre-dentine.

Methodology  RNA was isolated from dental pulp tissue and from cells retrieved by scraping the pre-dentine of freshly extracted human third molars (n = 15) using TRIzol^® reagent. Teeth were randomly assigned to the following temperature conditions: immediate RNA isolation after tooth extraction, liquid nitrogen (24 h), −80 °C (24 h), 20 °C (24 h) and 4 °C (6 h). RNA integrity was checked by the density of 28S and 18S ribosomal RNA. RT-PCR was used to analyse the expression of odontoblast makers (DSPP, DMP1 and MEPE) and the housekeeping gene GAPDH.

Results  All experimental conditions evaluated preserved RNA integrity. The three odontoblastic markers were amplified from the pulp tissue and from the cells associated with pre-dentine.

Conclusion  The four storage options allowed RNA isolation for RT-PCR analysis. These findings may facilitate the use of clinically derived human dental pulp and odontoblasts for endodontic research.

Aim  To compare the effectiveness of two rotary hybrid instrumentation techniques with focus on apical enlargement in molar teeth and to quantify and visualize spatial details of instrumentation efficacy in root canals of different complexity.

Methodology  Maxillary and mandibular molar teeth were scanned using X-ray microcomputed tomography. Root canals were prepared using either a GT/Profile protocol or a RaCe/NiTi protocol. Variables used for evaluation were the following: distance between root canal surfaces before and after preparation (distance after preparation, DAP), percentage of root canal area remaining unprepared and increase in canal volume after preparation. Root canals were classified according to size and complexity, and consequences of unprepared portions of narrow root canals and intraradicular connections/isthmuses were included in the analyses. One- and two-way anova were used in the statistical analyses.

Results  No difference was found between the two techniques: DAP_apical-third (P = 0.590), area unprepared_apical-third (P = 0.126) and volume increase_apical-third (P = 0.821). Unprepared root canal area became larger in relation to root canal size and complexity, irrespective of the technique used. Percentage of root canal area remaining unprepared was significantly lower in small root canals and complex systems compared to large root canals. The isthmus area per se contributed with a mean of 17.6%, and with a mean of 25.7%, when a narrow root canal remained unprepared.

Conclusions  The addition of isthmuses did not significantly alter the ratio of instrumented to unprepared areas at total root canal level. Distal and palatal root canals had the highest level of unprepared area irrespective of the two instrumentation techniques examined.

Aim  To investigate and determine possible associations of six tested bacteria belonging to ‘orange’ and ‘green’ complexes, in endo-periodontal lesions: Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga sputigena.

Methodology  Forty-six patients presenting with different types of endo-periodontal lesions were investigated. Clinical examinations, periapical radiographs and microbiological sampling from the canal system (endo) and periodontal pockets (perio) were performed. Qualitative and semiquantitative evaluation of bacteria was performed by polymerase chain reaction (PCR) and DNA–DNA hybridization (micro-IDent plus; Hain Lifescience, Germany).

Results  Extremely high bacterial loads in endodontic samples were recorded for P. micra, F. nucleatum and C. sputigena, while periodontal samples were often colonized by the same species, plus C. rectus. Significant association was recorded between F. nucleatum-endo and P. micra-endo (P = 0.03, Fisher’s exact test). There was marginal evidence of associations between: (i) C. sputigena-endo and C. sputigena-perio (P = 0.06, Fisher’s exact test); (ii) P. micra-endo and P. micra-perio (P = 0.05, Fisher’s exact test). Sensitivity to percussion was associated with an increased chance of cases with P. micra-endo (P = 0.03, Pearson chi-square test).

Conclusion  The findings suggest that F. nucleatum, P. micra and C. sputigena may play a role in the pathogenesis of endo-periodontal lesions.

Aim  To report several types of response of immature permanent teeth with infected necrotic pulp tissue and either apical periodontitis or abscess to revascularization procedures.

Methodology  Twenty immature permanent teeth with infected necrotic pulp tissue and either apical periodontitis or abscesses from 20 patients were included. The teeth were isolated with rubber dam, and pulp chambers was accessed through the crowns. The canals were gently irrigated with 5.25% sodium hypochlorite with minimal mechanical debridement. Calcium hydroxide was used as an inter-appointment intracanal medicament and placed into the coronal half of the canal space. After resolution of clinical signs and symptoms, bleeding was induced into the canal space from the periapical tissues using K-files. The coronal canal space was sealed with a mixture of mineral trioxide aggregate (MTA) and saline solution. The access cavity was filled with composite resin. These immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscesses were followed up from 6 to 26 months.

Results  Five types of responses of these immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures were observed: type 1, increased thickening of the canal walls and continued root maturation; type 2, no significant continuation of root development with the root apex becoming blunt and closed; type 3, continued root development with the apical foramen remaining open; type 4, severe calcification (obliteration) of the canal space; type 5, a hard tissue barrier formed in the canal between the coronal MTA plug and the root apex.

Conclusions  Based on this case series, the outcome of continued root development was not as predictable as increased thickening of the canal walls in human immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess after revascularization procedures. Continued root development of revascularized immature permanent necrotic teeth depends on whether the Hertwig’s epithelial root sheath survives in case of apical periodontitis/abscess. Severe pulp canal calcification (obliteration) by hard tissue formation might be a complication of internal replacement resorption or union between the intracanal hard tissue and the apical bone (ankylosis) in revascularized immature permanent necrotic teeth.