Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present vitro study was to examine the question whether devitalized Enterococcus faecalis (E. faecalis) cells can migrate into dentinal tubules and if that process takes place in a time-dependent manner. DESIGN: Sixty bovine root canals were incubated with devitalized and vital streptomycin-resistant E. faecalis strains after root canal enlargement (size 80, taper .02) with 3% NaOCl solution. Incubation times 7, 14, 21, 28, 35, and 42 days. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Statistical analysis was performed by Kruskal-Wallis (α = 0.05) and Mann-Whitney U test (p < 0.05). RESULTS: Devitalized E. faecalis strains were able to migrate into dentinal tubules. The total number and penetration depth of devitalized E. faecalis cells was lower compared to the vital suspension of E. faecalis. It was noted, that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. The migration took place in a time-dependent migration characteristic. CONCLUSIONS: Devitalized E. faecalis cells are still able to migrate into the dentinal tubules due to possible electrokinetic and osmotic processes. Thereby, increased exposure times lead to a time-dependent penetration characteristic. CLINICAL RELEVANCE: Since devitalized bacteria can migrate as well into dentinal tubules, the presence of bacteria within dentinal tubules cannot be interpreted as a failure of tested preparation regimens.

Effect of Sonic Application of Self-etch Adhesives on Bonding Fiber Posts to Root Canal Dentin.

PURPOSE: To evaluate the effect of sonic application of 5 different self-etch adhesives on the push-out bond strength of fiber posts in root canals. MATERIALS AND METHODS: In a preliminary test, 24 teeth were treated with manual and sonically assisted bonding, then a composite cylinder was built up to test the shear bond strength as a proof of principle. In the main test, 120 root canals were endodontically prepared and divided into 10 groups: 5 self-etch adhesives (Futurabond DC, Futurabond M, Futurabond U, Optibond XTR, Universalbond), each applied under manual and sonic application modes. After insertion of the fiber posts using the specific adhesive and a dual-curing composite, the teeth were sectioned and the push-out test was performed. The specimens were analyzed by light and scanning electron microscopy. Statistical analysis was performed using the Shapiro-Wilk test, one-way ANOVA and the Tamhane test. RESULTS: Sonic application of self-etch adhesive systems did not increase the bond strength of fiber posts in root canals. In general, the bond strength decreased from the coronal to the apical part of the root canal, irrespective of the applied method. The best post retention was achieved with Futurabond U and Optibond XTR. CONCLUSION: Sonic application of self-etch adhesives did not improve the fiber post retention in the root canal and can therefore not be recommended. Nevertheless, sonic application of etch-and-rinse adhesives can increase the bond strength to coronal dentin.

Application of Plant Extracts for the Prevention of Dental Erosion: An in situ/in vitro Study.

OBJECTIVES: Antiadherent and antibacterial effects of certain plant extracts have been proven to be beneficial in preventive dentistry. In the present in situ/in vitro crossover study, the impact of plant extracts rich in polyphenols on the erosion-protective properties of the in situ pellicle was evaluated. METHODS: Individual splints were prepared for 12 subjects for intraoral exposure of bovine enamel specimens. Following formation of a 1-min pellicle, watery plant extracts (leaves of the wild form of Ribes nigrum, the wild form of Origanum as well as a combination of both) were administered for 10 min in situ. Alternatively, a mouth rinse with fluorides (Elmex Kariesschutz) was performed for 1 min. After further oral exposure for 19/28 min, respectively, slabs were removed and incubated with HCl in vitro over 120 s (pH 2, 2.3, 3). The resulting calcium and phosphate release was quantified photometrically. Slabs with and without a 30-min in situ pellicle served as controls. The modification of pellicle ultrastructure was evaluated by transmission electron microscopy (TEM). RESULTS: Plant extracts modulated the erosion-protective properties of the native in situ pellicle in all test groups in a pH-dependent manner. The combination of R. nigrum leaves and Origanum enhanced the protective properties of the pellicle at all pH values; the administration of this preparation was comparable, yet superior, to the effect of the fluoridated mouth rinse. TEM images indicated that rinsing with R. nigrum leaves/Origanum yielded a distinctly thicker and more electron-dense pellicle. CONCLUSION: The combination of certain plant extracts offers a novel approach to the complementary prevention of dental erosion.

Quantification of Bacterial DNA from Infected Human Root Canals Using qPCR and DAPI after Disinfection with Established and Novel Irrigation Protocols.

The removal of bacterial infections within the root canal system is still a challenge. Therefore, the cleansing effect of established and new irrigation-protocols (IP) containing silver diamine fluoride (SDF) 3.8% on the whole root canal system was analyzed using quantitative PCR (qPCR) and 4',6-diamidino-phenylindole-(DAPI)-staining. Extracted human premolars were instrumented up to F2 (ProTaper Gold) under NaCl 0.9% irrigation and incubated with Enterococcus faecalis for 42 days. Subsequently, different ultrasonically agitated IP were applied to the roots: control (no irrigation), 1. NaOCl 3%, EDTA 20%, CHX 2%, 2. NaOCl 3%, EDTA 20%, 3. NaOCl 3%, EDTA 20%, SDF 3.8%, 4. SDF 3.8%, and 5. NaCl 0.9%. One half of the root was investigated fluorescent-microscopically with DAPI. The other half was grinded in a cryogenic mill and the bacterial DNA was quantified with qPCR. The qPCR results showed a statistically significant reduction of bacteria after the application of IP 1, 2, and 3 compared to the control group. While IP 4 lead to a bacterial reduction which was not significant, IP 5 showed no reduction. These data corresponded with DAPI staining. With qPCR a new molecular-biological method for the investigation of the complete root canal system was implemented. The novel IP 3 had an equally good cleansing effect as the already established IP.

An Automated Measurement Method for the Endodontic Working Width of Lower Molars by Means of Parametric Models Using Cone-beam Computed Tomographcy and Micro-Computed Tomography.

INTRODUCTION: A new method for the approximation of the root canal's cross-sectional shape and its working width using cone-beam computed tomographic (CBCT) or micro-computed tomographic (micro-CT) imaging was introduced. METHODS: Scanned data from 29 extracted human mandibular first and second molar distal root canals without instrumentation were reconstructed and analyzed with a self-developed measurement algorithm. The 3-dimensional volume models were sliced perpendicular to the vertical axis. Using different 2-dimensional parametric models, the contour of each root canal slice was approximated and used to determine the canal's cross-sectional dimensions. The measurements of minor width, major width, and the root canal's conicity were statistically analyzed using analysis of variance. RESULTS: The measured minor and major widths of the investigated root canals were significantly higher (probability value P < .05) when evaluated by CBCT images than the results obtained from micro-CT data. Both dimensions increased starting from the apical foramen (P < .01). The narrowest measured canal widths were 0.19-0.24 mm for CBCT imaging and 0.09-0.21 mm for micro-CT imaging in the apical part. The maximum values for conicity were between 13% and 17% in the cervical third. CONCLUSIONS: The 3-dimensional imaging data from CBCT and micro-CT imaging enabled a valuable anatomic assessment of the root canal's cross-sectional working width along the canal up to the physiological foramen in order to determine an adequate apical diameter as well as the correct measured taper in the cervical and medial part.

Mapping of the Micro-Mechanical Properties of Human Root Dentin by Means of Microindentation.

The extensive knowledge of root dentin's mechanical properties is necessary for the prediction of microstructural alterations and the teeth's deformations as well as their fracture behavior. Standardized microindentation tests were applied to apical, medial, and cervical root sections of a mandibular human first molar to determine the spatial distribution of the hard tissue's properties (indentation modulus, indentation hardness, Martens hardness, indentation creep). Using an indentation mapping approach, the inhomogeneity of mechanical properties in longitudinal as well as in transversal directions were measured. As a result, the tooth showed strongly inhomogeneous material properties, which depended on the longitudinal and transversal positions. In the transversal cutting planes of the cervical, medial, apical sections, the properties showed a comparable distribution. A statistical evaluation revealed an indentation modulus between 12.2 GPa and 17.8 GPa, indentation hardness between 0.4 GPa and 0.64 GPa and an indentation creep between 8.6% and 10.7%. The established standardized method is a starting point for further investigations concerning the intensive description of the inhomogeneous mechanical properties of human dentin and other types of dentin.

Quantification of Bacterial Colonization in Dental Hard Tissues Using Optimized Molecular Biological Methods.

Bacterial infections of root canals and the surrounding dental hard tissue are still a challenge due to biofilm formation as well as the complex root canal anatomy. However, current methods for analyzing biofilm formation, bacterial colonization of root canals and dental hard tissue [e.g., scanning electron microscopy, confocal laser scanning microscopy (CLSM) or determination of colony forming units (CFU)] are time-consuming and only offer a selective qualitative or semi-quantitative analysis. The aim of the present study is the establishment of optimized molecular biological methods for DNA-isolation and quantification of bacterial colonization via quantitative PCR (qPCR) from dental hard tissue. Root canals of human premolars were colonized with Enterococcus faecalis. For isolation of DNA, teeth were then grinded with a cryo mill. Since the hard tissues dentin and especially enamel belong to the hardest materials in the human organism, the isolation of bacterial DNA from root dentin is very challenging. Therefore, treatment steps for the isolation of DNA from grinded teeth were systematically analyzed to allow improved recovery of bacterial DNA from dental hard tissues. Starting with the disintegration of the peptidoglycan-layer of bacterial cells, different lysozyme solutions were tested for efficacy. Furthermore, incubation times and concentrations of chelating agents such as EDTA were optimized. These solutions are crucial for the disintegration of teeth and hence improve the accessibility of bacterial DNA. The final step was the determination of prior bacterial colonization of each root canal as determined by qPCR and comparing the results to alternative methods such as CFU. As a result of this study, optimized procedures for bacterial DNA-isolation from teeth were established, which result in an increased recovery rate of bacterial DNA. This method allows a non-selective and straightforward procedure to quantify bacterial colonization from dental hard tissue. It can be easily adapted for other study types such as microbiome studies and for comparable tissues like bones.

Continuous Measurement of Three-Dimensional Root Canal Curvature Using Cone-Beam Computed and Micro-Computed Tomography: A Comparative Study.

The knowledge of root canal curvature is crucial regarding the prevention of ledge formation, root perforation and the possibility of endodontic instruments' fracture during endodontic treatments. Therefore, a quantification method of the root canal curvature as well as the applicability of diagnostically relevant tomographic three-dimensional (3D) imaging data is necessary. Hereby, cone-beam computed tomography (CBCT) images and micro-computed tomography (µCT) data of distal root canals were analysed concerning the continuous three-dimensional curvature of human mandibular molars ( n = 50). The curvature of the canal's three-dimensional centre line was determined by evaluating the tomographic images. The centroids of each root canal slice were identified and approximated by spline curves to obtain the centre line and therefore, its curvature. Comparing the results evaluated from CBCT and µCT images, minimum radii of curvature of 2.6 mm and 2.1 mm were determined, respectively. The observation of the centre line demonstrated the requirement of the three-dimensional imaging data from CBCT and µCT for a reliable curvature analysis. Conclusively, the evaluation of CBCT and µCT images results in comparable radii of curvature. Thus, the application of the introduced method in combination with CBCT applied to patient cases could offer an important preliminary diagnostical step to prevent endodontic treatment complications.

Effects of Endodontic Irrigants on Material and Surface Properties of Biocompatible Thermoplastics.

Passive irrigation is an efficient method for a successful endodontic treatment. During sonic activation biocompatible polymer tips are used to activate irrigants. Compared to ultrasonic activation with metallic tips, polymer tips have the advantage of a reduced risk of fracture and minimise dentine damage. Hence, two polymers, polyether ether ketones (PEEK) and polyamide (PA6), were identified for the manufacturing of novel irrigation tips. The chemical resistance against the irrigants ethylenediaminetetraacetic acid (EDTA) 20%, chlorhexidine gluconate (CHX) 2% and sodium hypochlorite (NaOCl) 5.25% was analysed. Using microindentation, the change of hardness, elasticity, surface roughness and appearance of the polymers was determined. PA6 had a high absorption of irrigant compared to PEEK. PEEK was resistant to the investigated irrigants and showed no significant alteration of surface and mechanical properties, whereas PA6 slightly increased its hardness, elastic modulus and surface roughness during long-term exposure at 37 °C. However, PA6 tips seem to be a promising disposable product due to the material's high deformability and low manufacturing costs. Particularly with regard to structural-dynamic properties and high chemical resistance, PEEK can be considered as a material for reusable irrigation tips.

Odontoblast-like differentiation and mineral formation of pulpsphere derived cells on human root canal dentin in vitro.

BACKGROUND: The revitalization or regeneration of the dental pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell (DPC) spheres were applied to human root canal samples to evaluate their potential adoption for physiological tissue-like regeneration of the dental root canal by odontoblastic differentiation as well as cell-induced mineral formation. METHODS: DPC were cultivated into three-dimensional cell spheres and seeded on human root canal specimens. The evaluation of sphere formation, tissue-like behavior and differentiation as well as mineral formation of the cells was carried out with the aid of optical light microscopy, immunohistochemical staining and scanning electron microscopy (SEM). RESULTS: Spheres and cells migrated out of the spheres showed an intense cell-cell- and cell-dentin-contact with the formation of extra cellular matrix. In addition, the ingrowth of cell processes into dentinal tubules and the interaction of cell processes with the tubule walls were detected by SEM-imaging. Immunohistochemical staining of the odontoblast specific matrix proteins, dentin matrix protein-1, and dentin sialoprotein revealed an odontoblast-like cell differentiation in contact with the dentin surface. This differentiation was confirmed by SEM-imaging of cells with an odontoblast specific phenotype and cell induced mineral formation. CONCLUSIONS: The results of the present study reveal the high potential of pulp cells organized in spheres for dental tissue engineering. The odontoblast-like differentiation and the cell induced mineral formation display the possibility of a complete or partial "dentinal filling" of the root canal and the opportunity to combine this method with other current strategies.

An Approach for a Mathematical Description of Human Root Canals by Means of Elementary Parameters.

INTRODUCTION: Root canal geometry is an important factor for instrumentation and preparation of the canals. Curvature, length, shape, and ramifications need to be evaluated in advance to enhance the success of the treatment. Therefore, the present study aimed to design and realize a method for analyzing the geometric characteristics of human root canals. METHODS: Two extracted human lower molars were radiographed in the occlusal direction using micro-computed tomographic imaging. The 3-dimensional geometry of the root canals, calculated by a self-implemented image evaluation algorithm, was described by 3 different mathematical models: the elliptical model, the 1-circle model, and the 3-circle model. RESULTS: The different applied mathematical models obtained similar geometric properties depending on the parametric model used. Considering more complex root canals, the differences of the results increase because of the different adaptability and the better approximation of the geometry. CONCLUSIONS: With the presented approach, it is possible to estimate and compare the geometry of natural root canals. Therefore, the deviation of the canal can be assessed, which is important for the choice of taper of root canal instruments. Root canals with a nearly elliptical cross section are reasonably approximated by the elliptical model, whereas the 3-circle model obtains a good agreement for curved shapes.

Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. DESIGN: Thirteen root canals were incubated with devitalized (4days, 7days, 14days, 28days) and vital (28days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). RESULTS: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67µm±26.54µm after 7days, 230.67µm±111.5µm after 14days and 266.5µm±92.63µm after 28days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45µm) after 28days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. CONCLUSIONS: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.

Oral Health and Quality of Life in Old Age: A Cross-Sectional Pilot Project in Germany and Poland.

BACKGROUND: The process of ageing influences all dimensions of social life and personal well-being, but the influence of health on different dimensions of quality of life (QoL) among the elderly is rarely examined. OBJECTIVES: The aim of the pilot study is to test the feasibility of a comprehensive study design to evaluate general and dental health as well as QoL in a bi-national sample. In addition, this pilot study should allow for the exploration of potential interactions between QoL, socioeconomic, health and oral health variables. MATERIAL AND METHODS: Individuals aged 64 years and older (n = 100) from university dental clinics of the Wroclaw Medical University, Poland (n = 50) and of the University Hospital in Dresden, Germany (n = 50) were examined. The oral health status of participants was assessed by clinical examination. Socio-demographic, environmental and general health status were evaluated during the medical interview. General quality of life (GQoL) was assessed by an overall question with a visual analogue scale (VAS) from -5 (worst) to +5 (best). Health-related quality of life (HRQoL) and oral health-related quality of life (OHRQoL) were measured with the EQ-5D and OHIP-14 questionnaires. Statistical analyses comprised Pearson's c2 test, Wilcoxon test, linear regression model for statistical analysis and different multivariate linear regression analyses. RESULTS: For the GQoL-VAS-Score the results for QoL measurements were 1.22 ± 2.62 (x± SD), for EQ-5D-Score 7.45 ± 2.25 (x± SD), and for OHIP-14-ADD-Score 11.04 ± 13.56 (x± SD). Differences between Polish and German populations were observed. CONCLUSIONS: The study design proved to be feasible for a senior population. The overall GQoL question, EQ-5D and OHIP-14 were regarded as appropriate instruments. Subjective and objective (oral) health measures showed differences between Germany and Poland. For methodological reasons, these differences are not generalizable, but of value for study hypotheses in larger samples.

Possibilities and limits of imaging endodontic structures with CBCT.

An adequate portrayal of the root canal anatomy by diagnostic imaging is a prerequisite for a successful diagnosis and therapy in endodontics. The introduction of dental cone beam computed tomography (CBCT) has considerably expanded the scope of imaging diagnostics. The aim of the following study was to evaluate the imaging of endodontic structures with CBCT. One hundred and twenty teeth were examined with a CBCT device (ProMax 3D). Subsequently, the findings of the three-dimensional images were evaluated and compared to those of dental radiographs and tangential section preparations of the examined teeth. Results with high prevalence, such as existing roots and root canals, as well as results with low prevalence, e.g., extremely fine anatomical structures of the endodontic tissue, could be visualized precisely by dental CBCT; side canals, ramifications, communications, pulp stones, and obliterations could also be detected. Additionally, the length of curved root canals could be determined accurately. Likewise, root fractures were visualized reliably with CBCT. However, carious lesions could not be diagnosed adequately, and the evaluation of fillings and prosthetic restorations was complicated due to scattered X-ray artifacts. CBCT datasets qualify to visualize and diagnose small anatomical structures of the endodontic tissue.

Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro.

INTRODUCTION: Maintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration. METHODS: Human dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). RESULTS: Spheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation. CONCLUSIONS: The results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.