Seeing is believing? When scanning electron microscopy (SEM) meets clinical dentistry: The replica technique.

In restorative dentistry, the in situ replication of intra-oral situations, is based on a non-invasive and non-destructive scanning electron microscopy (SEM) evaluation method. The technique is suitable for investigation restorative materials and dental hard- and soft-tissues, and its interfaces. Surface characteristics, integrity of interfaces (margins), or fracture analysis (chipping, cracks, etc.) with reliable resolution and under high magnification (from ×50 to ×5,000). Overall the current study aims to share detailed and reproducible information about the replica technique. Specific goals are: (a) to describe detailed each step involved in producing a replica of an intra-oral situation, (b) to validate an integrated workflow based on a rational sequence from visual examination, to macrophotography and SEM analysis using the replica technique; (c) to present three clinical cases documented using the technique. A compilation of three clinical situations/cases were analyzed here by means the replica technique showing a wide range of possibilities that can be reached and explored with the described technique. This guidance document will contribute to a more accurate use of the replica technique and help researchers and clinicians to understand and identify issues related to restorative procedures under high magnification.

Evaluation of gap formation at the composite resin-tooth interface after using universal adhesives: In vitro SEM study using the replica technique.

The aim of this study was to evaluate gap formation after using two universal adhesives, Peak Universal Bond (P), which contains chlorhexidine and Adhese Universal (A), which contains 10-MDP monomer, and to compare them with two gold standard adhesives. The adhesives were tested in different etching modes, etch-and-rinse (ER) or self-etch (SE), and under thermomechanical loading (TML). Two identical Class V cavities were prepared in each of 30 human third molar teeth, and resin-composite restorations were made. Before and after TML, replicas of the restorations were prepared. Gap formation and their frequency according to the modified classification of Blunck and Zaslansky and the width of the maximum marginal gap (MG) were separately evaluated in enamel and dentin. Gap frequency did not differ significantly only in the enamel before TML, despite the use of different adhesives or etching modes. At both timepoints, the highest MG values were obtained with the Peak Universal Bond self-etch (PSE) group in enamel (6.2 µm before TML, 12.2 µm after TML) and the Peak Universal Bond etch-and-rinse (PER) group in dentine (21.3 µm before TML, 35.5 µm after TML). After TML, there was a significant increase of gap frequency for Adhese universal self-etch (ASE) group and MG values for PER, PSE, ASE groups at the resin-enamel interface, and gap frequency and MG values for all groups at the resin-dentin interface. Universal adhesive with 10-MDP monomer have beneficial effect on the resin-dentin interface, while chlorhexidine may reduce the resistance of the universal adhesive system to oral cavity conditions.

Sequential Replicas: Method for In Vivo Imaging of Plant Organ Surfaces that Undergo Deformation.

Complex geometry of plant organs and various types of organ surface deformation, including growth or hygroscopic movements, can be analyzed using sequential replica method. It enables obtaining a time-lapse series of high resolution images visualizing details of the examined surface and provides data sufficient for detailed computation of parameters characterizing surface deformation and geometry. Series of molds, made in dental polymer, representing the examined surface are used to obtain casts in epoxy resin or nail polish replicas, which are ready for microscopic examination, while the structure itself remains intact. Images obtained from the epoxy casts in scanning electron microscopy can be further used for 3D reconstruction and computation of local geometry. The sequential replica method is a universal method and can be applied to image complex shapes of a range of structures, like meristems, flowers, leaves, scarious bracts, or trichomes. Different plant species growing in various conditions can be studied.

3 D printed replica of the human temporal bone intended for teaching gross anatomy.

The anatomy of the human temporal bone is complex and, therefore, poses unique challenges for students. Furthermore, temporal bones are frequently damaged from handling in educational settings due to their inherent fragility. This report details the production of a durable physical replica of the adult human temporal bone, manufactured using 3D printing technology. The physical replica was printed from a highly accurate virtual 3D model generated from CT scans of an isolated temporal bone. Both the virtual and physical 3D models accurately reproduced the surface anatomy of the temporal bone. Therefore, virtual and physical 3D models of the temporal bone can be used for educational purposes in order to supplant the use of damaged or otherwise fragile human temporal bones.

Evaluation of adaptation of ceramic inlays using optical coherence tomography and replica technique.

Optical coherence tomography (OCT) has generally been used as a nondestructive technique to evaluate integrities of composite restorations. We investigated marginal and internal adaptations of ceramic inlay restorations with OCT and compared them to results with the silicone replica technique. Round-shaped class I cavities were prepared on 16 human maxillary first premolar teeth. Ceramic inlays were fabricated. Silicone replicas from inlays were obtained and sectioned to measure marginal and internal adaptations with a stereomicroscope (Leica Dfc 295, Bensheim, Germany). Inlays were cemented on respective teeth. Marginal and internal adaptations were then measured with the OCT system (Thorlabs, New Jersey, USA) in 200- µm intervals. Replica and OCT measurements were compared with independent samples t-tests. A paired t-test was used to evaluate the marginal and internal adaptations of each group (p < 0.05). Marginal and internal adaptations were 100.97 ± 31.36 and 113.94 ± 39.75 µm, respectively, using the replica technique and 28.97 ± 17.86 and 97.87 ± 21.83 µm, respectively, using OCT. The differences between the techniques were significant (p = 0.00 and p = 0.01, respectively). On evaluation within the groups, internal adaptation values were found to be significantly higher than the marginal adaptation values for the replica technique (p = 0.00) and OCT (p = 0.00). Therefore, the replica and OCT techniques showed different results, with higher values of marginal and internal adaptation found with the replica technique. Marginal and internal adaptation values of ceramic inlays, whether measured by replica or OCT techniques, were within clinically acceptable limits.

Reliability of light microscopy and a computer-assisted replica measurement technique for evaluating the fit of dental copings.

The aim of this in vitro study was to assess the reliability of two measurement systems for evaluating the marginal and internal fit of dental copings. Sixteen CAD/CAM titanium copings were produced for a prepared maxillary canine. To modify the CAD surface model using different parameters (data density; enlargement in different directions), varying fit was created. Five light-body silicone replicas representing the gap between the canine and the coping were made for each coping and for each measurement method: (1) light microscopy measurements (LMMs); and (2) computer-assisted measurements (CASMs) using an optical digitizing system. Two investigators independently measured the marginal and internal fit using both methods. The inter-rater reliability [intraclass correlation coefficient (ICC)] and agreement [Bland-Altman (bias) analyses]: mean of the differences (bias) between two measurements [the closer to zero the mean (bias) is, the higher the agreement between the two measurements] were calculated for several measurement points (marginal-distal, marginal-buccal, axial-buccal, incisal). For the LMM technique, one investigator repeated the measurements to determine repeatability (intra-rater reliability and agreement). For inter-rater reliability, the ICC was 0.848-0.998 for LMMs and 0.945-0.999 for CASMs, depending on the measurement point. Bland-Altman bias was -15.7 to 3.5 µm for LMMs and -3.0 to 1.9 µm for CASMs. For LMMs, the marginal-distal and marginal-buccal measurement points showed the lowest ICC (0.848/0.978) and the highest bias (-15.7 µm/-7.6 µm). With the intra-rater reliability and agreement (repeatability) for LMMs, the ICC was 0.970-0.998 and bias was -1.3 to 2.3 µm. LMMs showed lower interrater reliability and agreement at the marginal measurement points than CASMs, which indicates a more subjective influence with LMMs at these measurement points. The values, however, were still clinically acceptable. LMMs showed very high intra-rater reliability and agreement for all measurement points, indicating high repeatability.

Reliability of light microscopy and a computer-assisted replica measurement technique for evaluating the fit of dental copings

Abstract The aim of this in vitro study was to assess the reliability of two measurement systems for evaluating the marginal and internal fit of dental copings. Material and Methods: Sixteen CAD/CAM titanium copings were produced for a prepared maxillary canine. To modify the CAD surface model using different parameters (data density; enlargement in different directions), varying fit was created. Five light-body silicone replicas representing the gap between the canine and the coping were made for each coping and for each measurement method: (1) light microscopy measurements (LMMs); and (2) computer-assisted measurements (CASMs) using an optical digitizing system. Two investigators independently measured the marginal and internal fit using both methods. The inter-rater reliability [intraclass correlation coefficient (ICC)] and agreement [Bland-Altman (bias) analyses]: mean of the differences (bias) between two measurements [the closer to zero the mean (bias) is, the higher the agreement between the two measurements] were calculated for several measurement points (marginal-distal, marginal-buccal, axial-buccal, incisal). For the LMM technique, one investigator repeated the measurements to determine repeatability (intra-rater reliability and agreement). Results: For inter-rater reliability, the ICC was 0.848-0.998 for LMMs and 0.945-0.999 for CASMs, depending on the measurement point. Bland-Altman bias was −15.7 to 3.5 μm for LMMs and −3.0 to 1.9 μm for CASMs. For LMMs, the marginal-distal and marginal-buccal measurement points showed the lowest ICC (0.848/0.978) and the highest bias (-15.7 μm/-7.6 μm). With the intra-rater reliability and agreement (repeatability) for LMMs, the ICC was 0.970-0.998 and bias was −1.3 to 2.3 μm. Conclusion: LMMs showed lower interrater reliability and agreement at the marginal measurement points than CASMs, which indicates a more subjective influence with LMMs at these measurement points. The values, however, were still clinically acceptable. LMMs showed very high intra-rater reliability and agreement for all measurement points, indicating high repeatability.

Evaluation of adaptation of ceramic inlays using optical coherence tomography and replica technique

Abstract Optical coherence tomography (OCT) has generally been used as a nondestructive technique to evaluate integrities of composite restorations. We investigated marginal and internal adaptations of ceramic inlay restorations with OCT and compared them to results with the silicone replica technique. Round-shaped class I cavities were prepared on 16 human maxillary first premolar teeth. Ceramic inlays were fabricated. Silicone replicas from inlays were obtained and sectioned to measure marginal and internal adaptations with a stereomicroscope (Leica Dfc 295, Bensheim, Germany). Inlays were cemented on respective teeth. Marginal and internal adaptations were then measured with the OCT system (Thorlabs, New Jersey, USA) in 200- μm intervals. Replica and OCT measurements were compared with independent samples t-tests. A paired t-test was used to evaluate the marginal and internal adaptations of each group (p < 0.05). Marginal and internal adaptations were 100.97 ± 31.36 and 113.94 ± 39.75 μm, respectively, using the replica technique and 28.97 ± 17.86 and 97.87 ± 21.83 μm, respectively, using OCT. The differences between the techniques were significant (p = 0.00 and p = 0.01, respectively). On evaluation within the groups, internal adaptation values were found to be significantly higher than the marginal adaptation values for the replica technique (p = 0.00) and OCT (p = 0.00). Therefore, the replica and OCT techniques showed different results, with higher values of marginal and internal adaptation found with the replica technique. Marginal and internal adaptation values of ceramic inlays, whether measured by replica or OCT techniques, were within clinically acceptable limits.

¿Qué se entiende por extremidades y miembros?, ¿existe el reflejo axonal?. Réplica / What do we understand by extremities and limbs? Does the axonal reflex exist?. Reply

No disponible

The cast imaging of the osteon lacunar-canalicular system and the implications with functional models of intracanalicular flow.

A casting technique with methyl-methacrylate (MMA) was applied to the study of the osteon lacunar-canalicular network of human and rabbit cortical bone. The MMA monomer infiltration inside the vascular canals and from these into the lacunar-canalicular system was driven by capillarity, helped by evaporation and the resulting negative pressure in a system of small pipes. There was uniform, centrifugal penetration of the resin inside some osteons, but this was limited to a depth of four to five layers of lacunae. Moreover, not all of the osteon population was infiltrated. This failure can be the result of one of two factors: the incomplete removal of organic debris from the canal and canalicular systems, and lack of drainage at the osteon external border. These data suggest that each secondary osteon is a closed system with a peripheral barrier (represented by the reversal line). As the resin advances into the osteon, the air contained inside the canalicula is compressed and its pressure increases until infiltration is stopped. The casts gave a reliable visualization of the lacunar shape, position and connections between the lacunae without the need for manipulations such as cutting or sawing. Two systems of canalicula could be distinguished, the equatorial, which connected the lacunae (therefore the osteocytes) lying on the same concentric level, and the radial, which established connections between different levels. The equatorial canalicula radiated from the lacunar border forming ramifications on a planar surface around the lacuna, whereas the radial canalicula had a predominantly straight direction perpendicular to the equatorial plane. The mean length of the radial canalicula was 40.12 ± 10.26 µm in rabbits and 38.4 ± 7.35 µm in human osteons; their mean diameter was 174.4 ± 71.12 nm and 195.7 ± 79.58 nm, respectively. The mean equatorial canalicula diameter was 237 ± 66.04 nm in rabbit and 249.7 ± 73.78 nm in human bones, both significantly larger (P < 0.001) than the radial. There were no significant differences between the two species. The lacunar surface measured on the equatorial plane was higher in rabbit than in man, but the difference was not statistically significant. The cast of the lacunar-canalicular network obtained with the reported technique allows a direct, 3-D representation of the system architecture and illustrates how the connections between osteocytes are organized. The comparison with models derived by the assumption of the role of hydraulic conductance and other mechanistic functions provides descriptive, morphological data to the ongoing discussion on the Haversian system biology.

Calculation of bone graft volume using 3D reconstruction system

Objectives: To obtain bone “bioreplicas” for determining precisely the amount of biomaterial required for boneregeneration procedure. Study design: A case-control comparison with a total sample size of 20 cases, 10 controland 10 test samples. “Bioreplicas” were generated from helical CAT scans with 0.5 mm slices, without 3Dreconstruction or image filters. Bone defects in premolar and molar areas were treated with titanium mesh andxenograft (MP3) combined with resorbable carriers. Time taken to carry out procedures with and without the useof “bioreplicas” (in minutes), discrepancies between the grafts performed with and without “bioreplicas” and bonedefects (mm), and postoperative complications were registered. Results: No significant differences were recordedfor measurements of width and length of bone defect between patient bone and the biological models. Conclusions:The use of “bioreplicas” obtained by rapid prototyping is effective in treatment planning (AU)

High-resolution molecular localization by freeze-fracture replica labeling.

The freeze-fracture technique splits the frozen lipid bilayer membrane into two halves and immobilizes membrane proteins and lipids by the vacuum evaporation of platinum and carbon. After a treatment by SDS to remove extramembrane materials, the specimen is subjected to immunogold labeling, which gives information on the two-dimensional distribution of membrane molecules and their relationship to various differentiated structures. In combination with rapid freezing, the freeze-fracture technique has an advantage over other methods using conventional chemical fixation because the distribution of lipids as well as proteins can be observed at the mesoscale in a wide area of the membrane.

Replica exchange with solute tempering: efficiency in large scale systems.

We apply the recently developed replica exchange with solute tempering (REST) to three large solvated peptide systems: an alpha-helix, a beta-hairpin, and a TrpCage, with these peptides defined as the "central group". We find that our original implementation of REST is not always more efficient than the replica exchange method (REM). Specifically, we find that exchanges between folded (F) and unfolded (U) conformations with vastly different structural energies are greatly reduced by the nonappearance of the water self-interaction energy in the replica exchange acceptance probabilities. REST, however, is expected to remain useful for a large class of systems for which the energy gap between the two states is not large, such as weakly bound protein-ligand complexes. Alternatively, a shell of water molecules can be incorporated into the central group, as discussed in the original paper.

The structure of the Alzheimer amyloid beta 10-35 peptide probed through replica-exchange molecular dynamics simulations in explicit solvent.

The conformational states sampled by the Alzheimer amyloid beta (10-35) (Abeta 10-35) peptide were probed using replica-exchange molecular dynamics (REMD) simulations in explicit solvent. The Abeta 10-35 peptide is a fragment of the full-length Abeta 40/42 peptide that possesses many of the amyloidogenic properties of its full-length counterpart. Under physiological temperature and pressure, our simulations reveal that the Abeta 10-35 peptide does not possess a single unique folded state. Rather, this peptide exists as a mixture of collapsed globular states that remain in rapid dynamic equilibrium with each other. This conformational ensemble is dominated by random coil and bend structures with insignificant presence of an alpha-helical or beta-sheet structure. The 3D structure of Abeta 10-35 is seen to be defined by a salt bridge formed between the side-chains of K28 and D23. This salt bridge is also observed in Abeta fibrils and our simulations suggest that monomeric conformations of Abeta 10-35 contain pre-folded structural motifs that promote rapid aggregation of this peptide.

Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited.

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.

Técnica de replicação de amostras em positivo. Análise no Microscópio Eletrônico de Varredura / Positive replication tehcnique. Scanning Electron Microscope Assessment

A Microscopia Eletrônica de Varredura apresenta algumas desvantagens como a formação de trincas ou contração do dente durante seu preparo, assim como a impossibilidade de realizar observações in vivo. Para diminuir estes problemas, têm-se desenvolvido técnicas de replicação, avaliando a cópia e não o dente, o que permite estudar processos e seqüências de tratamento na mesma amostra, tanto in vitro como in vivo. Dessa forma, a proposição deste trabalho é avaliar morfologicamente a fidelidade de uma técnica de replicação, utilizando 2 resinas epóxicas e uma técnica de moldagem em dentes humanos extraídos. Uma vez eliminado o cemento no terço cervical radicular dos dentes com alta rotação, delimitou-se uma área de 4X4 na qual foi aplicado EDTA-T 24% por 3 minutos, seguindo da lavagem com soro e secagem com jato de ar, expondo túbulos dentinários. Estes seguimentos foram moldados, utilizando silicona de adição Aquasil (pesada e leve). Posteriormente, as moldagens foram vazadas com as resinas CMR 028-endurecedor CME-251 (Polipox, São Paulo) e o Epoxiglass 1504 e endurecedor 1603 (Epoxiglass-SP). Todas as réplicas e dentes naturais foram avaliados no MEV tomando fotomicrografias que foram avaliadas de acordo com o grau de fidelidade na réplica dos túbulos dentinários. Os resultados mostraram que a técnica de replicação reproduziu satisfatoriamente as características da superfície dentária.

Automation of a series of cutaneous topography measurements from silicon rubber replicas.

BACKGROUND/AIMS: Skin relief is a matter of interest for dermatologists and surgeons. One of the methods available for surface topography measurement is based on 3D profilometry using skin surface replicas. and most studies use statistical results obtained from a large number of skin replica samples. The advent of optical profilometers (without contact) made it possible to remove the solid positive replica and to reduce the duration of the profilometric data acquisition. Nevertheless this saving of time, to be really interesting, needs to automate the data acquisition on a series of negative replicas. METHODS/RESULTS: By adding a video camera to the optical profilometer and then by processing the resulting images, we have conceived a system able to carry out topographic measurements on a series of replicas loosely organized on a sample holder, without any human intervention. The silicon replicas in use have a very light colour: nearly white, sometimes slightly blue or green. The laser spot of the profilometer is so luminous that its red colour looks white through the camera. When choosing a replica holder with a matt dark colour and marking the left upper corner of the study area on the replica in black ink, the colours to be differentiated on the image are then close to the black one and the white one. We accordingly change the colour camera image into a black and white image (with 256 grey levels) and then carry out thresholdings to separate the different objects or information included in this image. With the use of a perfectly circular replica, of an accurately known size, laid on the sample holder at the center of the area filmed by the camera, we adjust the threshold level, which allows separation of the replica from its holder. We then move this calibrated replica in order to find the relationship between the size in pixels and the real size on the sample holder, in various positions of the video image. The software has four main built-in stages: Moving the sample holder beneath the sensor until a part of a replica is detected in the field of view of the camera; Moving the sample holder until this replica lies just in the middle of the image given by the camera; Recognition of the mark of the upper left corner of the surface area to be measured out inside this replica; and Moving the sample holder until the laser spot of the profilometer coincides with the origin of the surface area to be measured out, then carrying out this measurement. From the upper left corner of the sample holder, a scanning, line-by-line or column-by-column (according to the selected priority direction), is carried out until the successive replicas are found, and is stopped as soon as the number of replicas entered by the operator is reached. CONCLUSION: The simplicity of the algorithms used makes it possible to distinguish the next measurement area from the preceding one in a few seconds.