Back-scattered and secondary electron images of scanning electron microscopy in dentistry: a new method for surface analysis

Abstract

Objective. A scanning electron microscope (SEM) is a popular tool for investigating the root canal surface to visualize dentinal tubules, the smear layer and various root canal filling materials in endodontics. Most of the SEM micrographs taken in endodontic research are in secondary electrons (SE) mode, in which the topographic view of a subject can be demonstrated without giving any information about the real structure. Back-scattered electron (BSE) images are also used, which reveal some information about the internal structure while providing no topographic details. The aim of this study was to investigate the possibility of using back-scattered (BSE) and secondary electron (SE) mode of scanning electron microscopy (SEM) together for obtaining detailed information about biomaterials in relation to dental structures. Materials and methods. Mesiobuccal roots of four permanent maxillary molars were cleaned and shaped with rotary instruments. Two samples were obturated with gutta-percha and sealer. After 2 weeks, gutta-perch was removed using rotary instruments and chloroform. In the other phase of the study, white mineral trioxide aggregate was mixed and packed into five glass tubes and exposed to blood, deionized water, synthetic tissue fluid and egg white. All the samples were prepared for visualization under SE and BSE modes of SEM to observe the characteristics of material remnants and surface structures. Results. BSE mode illustrated different grey scale views which made it possible to differentiate dentin chips from filling material remnants on the surface of root canal dentin. In addition, SE mode focused on image topography, while a BSE detector showed new texture formation on the surface of white mineral trioxide aggregate exposed to proteinaceous fluids such as blood or egg white. Conclusions. Mapping BSE and SE micrographs helped us to better understand the structure of materials on the surface of root canal dentin and MTA. Moreover, analysis of structure of materials on the surface of root canal dentine and MTA can be performed better by mapping of BSE and SE micrographs.     

超声和手用器械龈下洁治后牙根面的扫描电镜观察

目的：通过超声和手用器械对牙周炎患牙根面处理的比较，来评价超声龈下洁治在牙周治疗中的作用。方法：选择１０个新鲜拔除的牙周炎患牙，随机分别用手用器械（第１组）和超声（第２组）洁治；选择４个临床诊断需拔除的牙周炎患牙分别行手用器械（第３组）和超声（第４组）龈下洁治，然后将患牙拔除。所有牙齿均在扫描电镜下观察。结果：超声龈下洁治能有效去除龈下菌斑及牙石，使根面较光滑平整，与手用器械洁治相比，无明显差异。结论：超声龈下洁治器是一种进行龈下洁治和根面平整的有效工具。     

Effectiveness of ozone against endodontopathogenic microorganisms in a root canal biofilm model

Aim To assess the antimicrobial efficacy of aqueous (1.25–20 μg mL^?1) and gaseous ozone (1–53 g m^?3) as an alternative antiseptic against endodontic pathogens in suspension and a biofilm model. Methodology Enterococcus faecalis, Candida albicans, Peptostreptococcus micros and Pseudomonas aeruginosa were grown in planctonic culture or in mono‐species biofilms in root canals for 3 weeks. Cultures were exposed to ozone, sodium hypochlorite (NaOCl; 5.25%, 2.25%), chlorhexidine digluconate (CHX; 2%), hydrogen peroxide (H₂O₂; 3%) and phosphate buffered saline (control) for 1 min and the remaining colony forming units counted. Ozone gas was applied to the biofilms in two experimental settings, resembling canal areas either difficult (setting 1) or easy (setting 2) to reach. Time‐course experiments up to 10 min were included. To compare the tested samples, data were analysed by one‐way anova . Results Concentrations of gaseous ozone down to 1 g m^?3 almost and aqueous ozone down to 5 μg mL^?1 completely eliminated the suspended microorganisms as did NaOCl and CHX. Hydrogen peroxide and lower aqueous ozone concentrations were less effective. Aqueous and gaseous ozone were dose‐ and strain‐dependently effective against the biofilm microorganisms. Total elimination was achieved by high‐concentrated ozone gas (setting 2) and by NaOCl after 1 min or a lower gas concentration (4 g m^?3) after at least 2.5 min. High‐concentrated aqueous ozone (20 μg mL^?1) and CHX almost completely eliminated the biofilm cells, whilst H₂O₂ was less effective. Conclusion High‐concentrated gaseous and aqueous ozone was dose‐, strain‐ and time‐dependently effective against the tested microorganisms in suspension and the biofilm test model.     

Single species biofilm-forming ability of root canal isolates on gutta-percha points

The participation of bacterial biofilms in the over-filled gutta-percha points associated with refractory periapical periodontitis has recently been reported. This study investigated the initial biofilm-forming ability of root canal isolates (Enterococcus faecalis, Streptococcus sanguis, Strep. intermedius, Strep. pyogenes, Staphylococcus aureus, Fusobacterium nucleatum, Propionibacterium acnes, Porphyromonas gingivalis and Prevotella intermedia) on gutta-percha points in vitro. Each bacterial strain was suspended in 100% cell culture medium or in culture medium containing 4.5, 45 or 90% (vol/vol) serum. The bacterial suspensions were then co-incubated anaerobically with gutta-percha points for 7 d. The gutta-percha points were processed for scanning electron microscopic observation and examined for biofilm presence and thickness. E. faecalis, Strep. sanguis, Strep. intermedius, Strep. pyogenes and Staph. aureus biofilms were generated on the surfaces of the specimens incubated in culture medium supplemented with 45 or 90% (vol/vol) serum. The E. faecalis and Strep. sanguis biofilms were significantly thicker than those of Strep. intermedius, Strep. pyogenes and Staph. aureus. No biofilms were detected on the specimens incubated with F. nucleatum, Prop. acnes, Porph. gingivalis and Prev. intermedia. These findings suggest that Gram-positive facultative anaerobes have the ability to colonize and form extracellular matrices on gutta-percha points, while serum plays a crucial role in biofilm formation.     

A scanning electron microscopic evaluation of root surfaces and the gutta-percha interface following root-end resection in vitro

AIM: The aim of this study was to evaluate the effects that various commonly used instruments have on root surface morphology, the obturating material, and the interface between the obturation and the root canal walls following root-end resection in vitro. METHODOLOGY: Sixty human single-rooted teeth with fully formed apices were collected and decoronated. The root canals were instrumented, and then obturated with thermoplasticized gutta-percha using AH-26 as the sealer. The roots were randomly divided into 12 different groups, and apical root-end resections were performed using eight different instrument configurations, and two different directions in which the bur moved across the root face in relation to its direction of rotation. Epoxy resin replicas of the resected root ends were examined using scanning electron microscopy. RESULTS: Each instrument produced a characteristic surface finish on the resected root end that mirrored its cutting profile. Irrespective of the design of the bur used, smearing and shredding of the gutta-percha across the root face occurred only when the handpiece was moved across the root face in reverse direction in relation to the direction of rotation of the bur. CONCLUSIONS: To ensure minimal disruption and distortion of the root filling and to prevent shredding of the gutta-percha interface, care should be taken to ensure that the final pass of the bur across the root canal is in the correct direction in relation to its direction of rotation.     

Cleaning efficacy using two engine-driven systems versus manual instrumentation in curved root canals: a scanning electron microscopic study

Introduction

This ex vivo study evaluated the cleanliness of curved root canal walls after chemomechanical instrumentation using two automated systems versus manual instrumentation while using a standardized irrigation protocol.

Methods

Thirty mesial root canals of extracted human first and second mandibular molars were prepared with the TiLOS hybrid engine-driven instrumentation system (Ultradent Products Inc, South Jordan, UT) (n = 10), ProTaper engine-driven file series (n = 10), and manual instrumentation (n = 10). Irrigation was performed using alternately 5.25% sodium hypochlorite and 17% EDTA followed by rinsing with distilled water. After the roots were split longitudinally, the presence of debris and/or smear layer was visualized using serial scanning electron microscopic digital photomicrographs taken at 1, 5, and 10 mm from the working length. Mean scores for debris and the smear layer were calculated and statistically analyzed for significance (P < .05) between groups using the Kruskal-Wallis nonparametric analysis of variance and Dunn tests. The data obtained at each evaluation level for each group were analyzed using the Friedman and Tukey multiple comparison tests.

Results

No significant differences (P > .05) were found between TiLOS and ProTaper (Dentsply/Tulsa Dental, Tulsa, OK) groups, whereas both performed significantly better than the manual instrumentation group.

Conclusions

Engine-driven TiLOS and ProTaper instrumentation systems combined with a standardized irrigation protocol produced cleaner root canal walls than the manual instrumentation technique although complete cleanliness was not achieved.     

Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota

Sánchez MC, Llama‐Palacios A, Blanc V, León R, Herrera D, Sanz M. Structure, viability and bacterial kinetics of an in vitro biofilm model using six bacteria from the subgingival microbiota. J Periodont Res 2011; 46: 252–260. © 2011 John Wiley & Sons A/S Background and Objective: There are few in vitro models available in the scientific literature for study of the structure, formation and development of the subgingival biofilm. The purpose of this study was to develop and validate an in vitro biofilm model, using representative selected bacteria from the subgingival microbiota. Material and Methods: Six standard reference strains were used to develop biofilms over sterile ceramic calcium hydroxyapatite discs coated with saliva within the wells of presterilized polystyrene tissue culture plates. The selected species represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The structure of the biofilm obtained was studied using a vital fluorescence technique in conjunction with confocal laser scanning microscopy. The biofilm bacterial kinetics were studied by terminal restriction fragment length polymorphism analysis. Results: After 12 h, initial and early colonizers were the first microorganisms detected adhering to the calcium hydroxyapatite discs. The intermediate colonizer F. nucleatum was not detected in the model until 24 h of incubation. Late colonizers A. actinomycetemcomitans and P. gingivalis could be measured inside the biofilm after 48 h. The biofilm reached its steady state between 72 and 96 h after inoculation, with bacterial vitality increasing from the hydroxyapatite surface to the central part of the biofilm. Conclusion: An in vitro biofilm model was developed and validated, demonstrating a pattern of bacterial colonization and maturation similar to the in vivo development of the subgingival biofilm.     

龋齿、牙周炎患牙和健康牙的菌斑生物膜特征

目的研究牙面菌斑生物膜特征与口腔疾病的关系。方法选择牙周健康而牙冠严重龋坏的龋齿5颗、无龋损而极度松动的牙周炎患牙6颗及正畸原因拔除的健康牙4颗,在扫描电镜下,观察分析龈上、龈下及移行生态区的菌斑生物膜特征。结果龋齿、牙周炎患牙和健康牙的牙面均观察到细菌混合物组成的菌斑生物膜,健康牙菌斑生物膜以球菌为主,放线菌和短杆菌少量;龋齿牙的龋坏处为坏死组织和细菌,龋边缘及龈沟处的球菌和短杆菌较健康牙多;牙周炎患者牙菌斑生物膜的细菌种类多,在龈上、龈下移行处可见典型的玉米棒状菌斑或以杆菌为主的紧密附着菌斑,龈下可见球菌、杆菌、梭菌及螺旋体等构成的复杂菌斑。结论龋齿、牙周炎患牙和健康牙菌斑生物膜细菌组成、集聚秩序和立体结构不同,菌斑生物膜的形成与细菌的附着、集聚、生长有关,也与局部病变密切相关。     

Deformation and fracture of RaCe and K3 endodontic instruments according to the number of uses

AIM: To evaluate, by scanning electron microscopy, the deformation and fracture of NiTi RaCe and K3 size 25, 0.04 taper instruments. METHODOLOGY: Ten sets of instruments from RaCe and K3 NiTi rotary systems were used to prepare 100 simulated canals in epoxy resin blocks with 20 or 40 degree curvatures beginning 8 or 12 mm from the orifice. Each instrument set was used to prepare five simulated canals using a crowndown technique. The size 25, 0.04 taper instruments were analysed by SEM when new and again after each use. Three observers scored images of the instruments after each use for distortion of the spirals (no distortion, distortion of one spiral or distortion of more than one spiral), wear (no wear, small, moderate or severe wear) and fracture (yes or no). Two-way anova was used to analyse differences between instruments for distortion and wear; Fisher's exact test looked for differences related to fracture of instruments. RESULTS: No fractures occurred with K3 instruments, whereas six RaCe instruments fractured (P = 0.005). A statistically significant difference occurred between RaCe and K3 instruments in terms of distortion of spirals and surface wear (P < 0.001). Distortion of spirals and wear increased with progressive use of RaCe instruments, whereas K3 instruments remained relatively undamaged after their fifth use. The simulated canals with smaller radii of curvature were positively associated with fracture of RaCe instruments. CONCLUSIONS: A significant difference was found between RaCe and K3 in terms of deformation and fracture of size 25, 0.04 taper instruments; K3 instruments had more favourable results.     

Behavior of human periodontal ligament cells on CO2 laser irradiated dentinal root surfaces: an in vitro study

OBJECTIVE: The aim of this study was to investigate the in vitro attachment behavior of human periodontal ligament fibroblasts on periodontally involved root surface after conditioning with CO2 laser and to compare its efficacy with chemical conditioning agents, namely tetracycline hydrochloride, citric acid, hydrogen peroxide (H2O2) and EDTA, using scanning electron microscopy. METHODS: A total of 84 scaled and root-planed specimens from periodontally involved single-rooted human teeth showing hopeless prognosis were selected and assigned to two groups. One group was lased with a CO2 laser (from 5 cm at 3 W for 0.8, 1.0 and 1.2 s), and the other group was treated with either tetracycline hydrochloride (2.5%), citric acid (saturated solution, pH 1), H2O2 (6%) or EDTA (5%; pH 7.4) for 3 min. The specimens were then seeded with human periodontal ligament fibroblasts, incubated for either 12 h or 24 h, and then the cell attachment behavior was observed. RESULTS: CO2 laser irradiation for 1.0 s was found to be the most efficient, showing consistently good cell attachment with the highest mean value (15.00 +/- 3.41 cells/10,000 microm2 after incubation for 12 h and 29.17 +/- 2.04 cells/10,000 microm2 after 24 h), followed by irradiation for 0.8 s (13.11 +/- 3.04 cells/10,000 microm2 after incubation for 12 h and 22.91 +/- 7.10 cells/10,000 microm2 after 24 h). Charring was observed following irradiation for 1.2 s. Amongst chemical conditioning agents, citric acid was found to be the most efficient, with a mean cell attachment of 17.82 +/- 2.16 cells/10,000 microm2 after incubation for 12 h and 23.62 +/- 1.94 cells/10,000 microm2 after 24 h. EDTA and H2O2 did not do well in the study. CONCLUSION: The results suggest that CO2 laser irradiation for 1.0 s may promote comparatively better attachment of periodontal ligament fibroblast on dentinal root surfaces than the conventional chemical conditioning agents used in the study.     

Microbial adhesion and viability on novel CAD/CAM framework materials for implant-supported hybrid prostheses

The aim of the study was to investigate the adhesion and viability of Streptococcus oralis and Candida albicans under in vitro conditions on CAD/CAM framework materials for implant-supported hybrid prostheses. Twenty-nine specimens were prepared from each of three different materials: ZR (zirconia), PEEK (polyether ether ketone) and CoCr₄ (CoCr₄ alloy). The experimental part included surface roughness (SR) and contact angle of water (CAW) measurements, followed by colony forming unit (CFU), cell viability assay and scanning electron microscopy (SEM) analyses of Strep. oralis and C. albicans biofilms on the materials’ surfaces. Kruskal-Wallis and one-way analysis of variance (ANOVA) tests were used for differences between materials, and the correlation between measurements was estimated using Spearman's correlation coefficient. PEEK specimens revealed higher SR, CAW and CFU mean values, than ZR and CoCr₄ specimens. Strong positive correlation was found between SR and CFU and between CAW and CFU for both microbial species. Cell viability assay revealed similar values for both species across materials. Higher numbers of Strep. oralis and C. albicans on PEEK specimens confirm the impact of the higher surface roughness and contact angle values on the microbial adhesion and describes PEEK as less desirable than ZR and CoCr₄ from microbiological aspect.     

Subgingival debridement of root surfaces with a micro-brush: macroscopic and ultrastructural assessment

AIM: The aim of this study was to assess the use of a micro-brush to remove plaque deposits from subgingival, periodontally involved root surfaces in vivo. METHODS: 30 periodontally involved teeth requiring extraction for periodontal or prosthetic reasons in 26 adult patients were utilised. For inclusion, teeth had to display at least 30% bone loss radiographically. Following the establishment of local anaesthesia, grooves were cut on the proximal root surface adjacent to the gingival margin at the line angles. For each tooth, 1 proximal root surface was rubbed with the micro-brush for 2 min to the depth of the pocket whilst the other root surface acted as an undebrided control. The teeth were then extracted, rinsed in 0.85% NaCl, stained with 2% erythrosine solution and photographed. The amount of erythrosine staining on each subgingival, periodontally involved root surface was assessed by tracing the areas of stain on a colour photograph and scanning the tracings into a computerised image tracing program. RESULTS: Results were expressed as the % of the periodontally involved root-surface area that exhibited staining. Stained areas were further examined with the scanning electron microscope (SEM). The undebrided root surfaces each displayed 100% staining. The debrided surfaces (with probing pocket depths of 4-10 mm) displayed mean staining of 16.1% (SD +/-7.1%) of the proximal surface area. SEM assessment showed that undebrided root surfaces were covered with thick deposits of bacteria. On debrided surfaces, stain-free areas were free of plaque whilst areas of faint staining exhibited either no plaque, calculus deposits or scanty, isolated islands of bacteria. Bacteria had been partially removed from the surface of calculus in some areas. CONCLUSIONS: The findings indicate that subgingival debridement with a micro-brush is effective in removing plaque deposits from periodontally involved root surfaces.     

Scanning electron microscopy of attrited dentinal surfaces and subjacent dentin in human teeth

abstract – Attrited dentinal surfaces and subjacent dentin were examined in the scanning electron microscope. In a central area of the exposed surfaces the dentinal tubules were occluded, whereas peripherally most tubules were open. Deposits, apparently dental plaque, were observed on the exposed dentin as uneven, but often continuous, layers covering whole areas of the surfaces. Loss of dentin within delimited areas of the surfaces was observed. The resulting defects could hardly be related to attrition, and it seemed that they were caused by erosive or caries-like processes. The dentin subjacent to the attrited surfaces was characterized by mineralized inclusions in the dentinal tubules. The inclusions consisted of irregularly rod-shaped crystals forming a meshwork on the tubule walls. Other crystals were formed as prisms, seemingly rhombohedrons. Crystals which were hexagonal in cross-section were also seen. The exposure of the dentin also seemed to have influence on the organic content of the tubules. Thus, it appeared that ground substance was removed from the intratubular fibers permitting the observation of cross-banding typical of collagen at their surfaces. In some tubules the fibers seemed to be covered by mineral.     

自然牙菌斑生物膜模型的构建及应用

目的:构建人体内牙菌斑生物膜模型,观察不同时间自然形成菌斑生物膜的特点,体外观察精油成分漱口液对生物膜的渗透及杀菌效果。方法:按纳入标准选取6名志愿者,将刻有凹槽的羟基磷灰石圆片固定于活动夹板,配戴夹板,分别于6、24、48h取出圆片,于激光共聚焦显微镜下观察生物膜结构,检测总生物膜厚度,以及其中活菌厚度(μm)和荧光强度百分比(%)。观察各个时期形成的生物膜经漱口液作用1、5、15和30min后生物膜中活菌厚度及荧光强度的变化。应用SPSS13.0软件包进行随机区组单因素方差分析,SNK法进行均数两两比较。结果:6、24、48h形成的菌斑生物膜厚度分别为(11.92±4.63)μm、(18.63±4.66)μm和(27.55±6.35)μm,48h内菌斑生物膜逐渐增厚(P<0.05),尤以6h内增厚速度最快。不同时间形成的菌斑生物膜中,活菌厚度、荧光强度无显著差异(P>0.05)。6h形成的生物膜经漱口液作用,5min后活菌厚度和荧光强度即明显降低,15min后降至最低(P<0.01);24h和48h的生物膜则在漱口液作用15min后出现活菌厚度和荧光强度明显降低(P<0.05)。结论:本实验建立了体内收集...