Effect of data binning and frame averaging for micro-CT image acquisition on the morphometric outcome of bone repair assessment.

Despite the current advances in micro-CT analysis, the influence of some image acquisition parameters on the morphometric assessment outcome have not been fully elucidated. The aim of this study was to determine whether data binning and frame averaging affect the morphometric outcome of bone repair assessment using micro-CT. Four Wistar rats' tibiae with a surgically created bone defect were imaged with micro-CT six times each, frame averaging set to 1 and 2, and data binning set to 1, 2 and 4, for each of the averaging values. Two-way ANOVA followed by Bonferroni tests assessed the significance of frame averaging and data binning on a set of morphometric parameters assessed in the image volumes (p < 0.01). The effect of frame averaging was not significant for any of the assessed parameters. Increased data binning led to larger trabecular thickness. In contrast, smaller bone volume fraction and bone volume were found as data binning increased. Trabeculae number and trabecular separation were not influenced by any of the parameters. In conclusion, the morphometric outcome of bone repair assessment in micro-CT demonstrated dependency upon data binning, but not frame averaging. Therefore, image acquisition of small anatomical structures (e.g., rat trabeculae) should be performed without data binning.

Micro-CT data of early physiological cancellous bone formation in the lumbar spine of female C57BL/6 mice.

Micro-CT provides critical data for musculoskeletal research, yielding three-dimensional datasets containing distributions of mineral density. Using high-resolution scans, we quantified changes in the fine architecture of bone in the spine of young mice. This data is made available as a reference to physiological cancellous bone growth. The scans (n = 19) depict the extensive structural changes typical for female C57BL/6 mice pups, aged 1-, 3-, 7-, 10- and 14-days post-partum, as they attain the mature geometry. We reveal the micro-morphology down to individual trabeculae in the spine that follow phases of mineral-tissue rearrangement in the growing lumbar vertebra on a micrometer length scale. Phantom data is provided to facilitate mineral density calibration. Conventional histomorphometry matched with our micro-CT data on selected samples confirms the validity and accuracy of our 3D scans. The data may thus serve as a reference for modeling normal bone growth and can be used to benchmark other experiments assessing the effects of biomaterials, tissue growth, healing, and regeneration.

Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography.

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm-1) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

Standardization of Microcomputed Tomography for Tracheal Tissue Engineering Analysis.

Tracheal tissue engineering has become an active area of interest among clinical and scientific communities; however, methods to evaluate success of in vivo tissue-engineered solutions remain primarily qualitative. These evaluation methods have generally relied on the use of photographs to qualitatively demonstrate tracheal patency, endoscopy to image healing over time, and histology to determine the quality of the regenerated extracellular matrix. Although those generally qualitative methods are valuable, they alone may be insufficient. Therefore, to quantitatively assess tracheal regeneration, we recommend the inclusion of microcomputed tomography (µCT) to quantify tracheal patency as a standard outcome analysis. To establish a standard of practice for quantitative µCT assessment for tracheal tissue engineering, we recommend selecting a constant length to quantify airway volume. Dividing airway volumes by a constant length provides an average cross-sectional area for comparing groups. We caution against selecting a length that is unjustifiably large, which may result in artificially inflating the average cross-sectional area and thereby diminishing the ability to detect actual differences between a test group and a healthy control. Therefore, we recommend selecting a length for µCT assessment that corresponds to the length of the defect region. We further recommend quantifying the minimum cross-sectional area, which does not depend on the length, but has functional implications for breathing. We present empirical data to elucidate the rationale for these recommendations. These empirical data may at first glance appear as expected and unsurprising. However, these standard methods for performing µCT and presentation of results do not yet exist in the literature, and are necessary to improve reporting within the field. Quantitative analyses will better enable comparisons between future publications within the tracheal tissue engineering community and empower a more rigorous assessment of results. Impact statement The current study argues for the standardization of microcomputed tomography (µCT) as a quantitative method for evaluating tracheal tissue-engineered solutions in vivo or ex vivo. The field of tracheal tissue engineering has generally relied on the use of qualitative methods for determining tracheal patency. A standardized quantitative evaluation method currently does not exist. The standardization of µCT for evaluation of in vivo studies would enable a more robust characterization and allow comparisons between groups within the field. The impact of standardized methods within the tracheal tissue engineering field as presented in the current study would greatly improve the quality of published work.

Investigation on the validity of 3D micro-CT imaging in the fish brain.

BACKGROUND: Micro-computed tomography (CT) is a non-invasive technique that is used to obtain three-dimensional (3D) images of tissue structure in small animals. Compared with extensive mammal studies, few 3D imaging studies of fish have been conducted using micro-CT. An optimized method for imaging fish tissue structure is necessary, because they have adapted to diverse environments via functional and structural specialization. NEW METHOD: Brains of three species with different sizes and habitats were fixed in 4% paraformaldehyde and immersed in non-ionic iodinated contrast agent (Iopamiron). We examined the relationship between Iopamiron concentration and immersion time to determine universally optimal conditions for use in fish. RESULTS: We reconstructed 3D images of whole fish brains from cross-sections of brains from the Malabar grouper (Epinephelus malabaricus), bastard halibut (Paralichthys olivaceus), and threespot wrasse (Halichoeres trimaculatus). Developmental changes in brain structure were observed in the bastard halibut. Most brain regions of the threespot wrasse were distinguishable, although inner regions of the brain were less visible. COMPARISON WITH EXISTING METHODS: Histological techniques are typically used to observe fish brain structure, despite its drawbacks in terms of tissue sample preparation (shrinkage and distortion) and image capture (3D image constriction). The technique examined in the present study solves these problems and allows for the simultaneous handling of multiple specimens. CONCLUSION: Micro-CT imaging is suitable for observing the surfaces and inner structures of fish of various species.

Microcomputed Tomography Calibration Using Polymers and Minerals for Enamel Mineral Content Quantitation.

OBJECTIVE: The aim of this paper was to develop calibration standards (CSs) that are readily available for clinical researchers for the quantitation of enamel mineral content. METHOD: Polyethylene terephthalate (PET), acetal, polyphenylene sulfide (PPS), selenite, Egyptian alabaster, aragonite, and fluorite were fashioned into discs, and their densities were measured and stacked for microcomputed tomography examination. Frame averaging, flat-field correction, pre-filtration, and beam-hardening correction were applied. CSs were checked for homogeneity. The linear relationship between the mean greyscale value (GSV) of each disc and its physically calculated density was explored, and reproducibility was tested. A calibration function was established and then validated using a bovine enamel disc and sound enamel of extracted human premolar teeth. RESULTS: Measured densities were PET (ρ = 1.38 g/cm3), acetal (ρ = 1.41 g/cm3), PPS (ρ = 1.64 g/cm3), selenite (ρ = 2.24 g/cm3), Egyptian alabaster (ρ = 2.7 g/cm3), aragonite (ρ = 2.72 g/cm3), and fluorite (ρ = 3.11 g/cm3). Examination of the profile sections of CSs confirmed the uniformity of GSVs with minimal beam-hardening effect. A squared Pearson correlation coefficient of R2 = 0.994 was determined between the mean GSV of each CS and its calculated density and was reproduced at different settings with R2 >0.99. A linear regression equation of density (y) versus GSV (x) was established using the least squares regression equation method. The estimated density of the bovine enamel disc (2.48 g/cm3) showed high accuracy when compared to the physically measured value (2.45 g/cm3). The -relative error was 1.2%. Densities of sound enamel in the extracted human premolar teeth were 2.6-3.1 g/cm3. CONCLUSIONS: This is a simple, valid, and reproducible method to quantitate enamel mineral content. This simple, yet accurate system could be used to expand knowledge in the field of enamel caries research.

Ultrasonic cartilage thickness measurement is accurate, reproducible, and reliable-validation study using contrast-enhanced micro-CT.

BACKGROUND: Ultrasonography is a fast and patient-friendly modality to assess cartilage thickness. However, inconsistent results regarding accuracy have been reported. Therefore, we asked what are (1) the accuracy, (2) reproducibility, and (3) reliability of ultrasonographic cartilage thickness measurement using contrast-enhanced micro-CT for validation? METHODS: A series of 50 cartilage-bone plugs were harvested from fresh bovine and porcine joints. Ultrasonic cartilage thickness was determined using an A-mode, 20-MHz hand-held ultrasonic probe with native (1580 m/s) and adjusted speed of sound (1696 m/s). All measurements were performed by two observers at two different occasions. Angle of insonation was controlled by tilting the device and recording minimal thickness. Retrieval of exact location for measurement was facilitated by aligning the circular design of both cartilage-bone plug and ultrasonic device. There was no soft tissue interference between cartilage surface and ultrasonic probe. Ground truth measurement was performed using micro-CT with iodine contrast agent and a voxel size of 16 µm. The mean cartilage thickness was 1.383 ± 0.402 mm (range, 0.588-2.460 mm). RESULTS: Mean accuracy was 0.074 ± 0.061 mm (0.002-0.256 mm) for native and 0.093 ± 0.098 mm (0.000-0.401 mm) for adjusted speed of sound. Bland-Altman analysis showed no systematic error. High correlation was found for native and adjusted speed of sound with contrast-enhanced micro-CT (both r = 0.973; p < 0.001). A perfect agreement for reproducibility (intraclass correlation coefficient [ICC] 0.992 and 0.994) and reliability (ICC 0.993, 95% confidence interval 0.990-0.995) was found. CONCLUSIONS: Ultrasonic cartilage thickness measurement could be shown to be highly accurate, reliable, and reproducible. The A-mode ultrasonic cartilage thickness measurement is a fast and patient-friendly modality which can detect early joint degeneration and facilitate decision making in joint preserving surgery.

Uncertainties of synchrotron microCT-based digital volume correlation bone strain measurements under simulated deformation.

Digital Volume Correlation (DVC) is used to measure internal displacements and strains in bone. Recent studies have shown that Synchrotron radiation micro-computed tomography (SR-microCT) can improve the accuracy and precision of DVC. However, only zero-strain or virtually-moved test have been used to quantify the DVC uncertainties, leading to potential underestimation of the measurement errors. In this study, for the first time, the uncertainties of a global DVC approach have been evaluated on repeated SR-microCT scans of bovine cortical bone (voxel size: 1.6 µm), which were virtually deformed for different magnitudes and along different directions. The results showed that systematic and random errors of the normal strain components along the deformation direction were higher than the errors along unstrained directions. The systematic percentage errors were smaller for larger virtual deformations. The random percentage error was in the order of 10% of the virtual deformation. However, higher errors were localized at the boundary of the volumes of interest, perpendicular to the deformation direction. When only the central region of the samples was considered (100 µm layers removed from the borders where the deformation was applied), the errors in the direction of virtual deformation were comparable to the errors in the unstrained directions. In conclusion, the method presented to estimate the uncertainties of DVC is suitable for testing anisotropic specimens as cortical bone. The good agreement between the uncertainties in measurements of strain components obtained with this approach and with the simpler zero-strain-test suggests that the latter is adequate in the tested deformation scenarios.

Evaluation of Threshold Values for Root Canal Filling Voids in Micro-CT and Nano-CT Images.

While several materials and techniques have been used to assess the quality of root canal fillings in micro-CT images, the lack of standardization in scanning protocols has produced conflicting results. Hence, the aim of this study was to determine a cutoff voxel size value for the assessment of root canal filling voids in micro-CT and nano-CT images. Twenty freshly extracted mandibular central incisors were used. Root canals were prepared with nickel titanium files to an ISO size 40/0.06 taper and then filled with a single cone (40/0.06 taper) and AH Plus sealer. The teeth were scanned with different voxel sizes with either micro-CT (5.2, 8.1, 11.2, and 16.73 µm) or nano-CT (1.5 and 5.0 µm) equipment. Images were reconstructed and analyzed with the NRecon and CTAn software. Void proportion and void volume were calculated for each tooth in the apical, middle, and coronal thirds of the root canal. Kruskal-Wallis and post hoc Mann-Whitney U tests were performed with a significance level of 5%. In micro-CT images, significantly different results were detected among the tested voxel sizes for void proportion and void volume, whereas no such differences were found in nano-CT images (p > 0.05). Micro-CT images showed higher void numbers over the entire root length, with statistically significant differences between the voxel size of 16.73 µm and the other sizes (p < 0.05). The values of the different nano-CT voxel sizes did not significantly differ from those of the micro-CT (5.2, 8.1, and 11.2 µm), except for the voxel size of 16.73 µm (p < 0.05). All tested voxel sizes enabled the detection of root canal filling voids except for the voxel size of 16.73 µm. Bearing in mind the limitations of this study, it seems that a voxel size of 11.2 µm can be used as a reliable cutoff value for the assessment of root canal filling voids in micro-CT imaging.

Quantification of voxel values in micro computed tomography using multiple porosity hydroxyapatite blocks.

Microfocus X-ray computed tomography (micro-CT) has been applied as a method for the nondestructive and detailed assessment of trabecular bone patterns and tooth structure. Voxel values obtained from micro-CT are not absolute values. Therefore, voxel values were assessed using hydroxyapatite (HA) blocks with a different vesicle rate to quantify voxel values of micro-CT images in the present investigation.HA blocks with 4 levels of porosity and a block with a soft tissue-equivalent density were used, and the voxel values of each block were measured. Correlations between voxel values of micro-CT and HA densities were analyzed. Also, black and white binary images were produced, and the ratios of white pixels to pixels in regions of interest (ROIs) were calculated. The relationship between voxel values of micro-CT and HA densities could be regressed using a linear equation, and the correlation coefficient was high. Also, there were no significant differences in the regression equations between the first and second times. Voxel values of micro-CT might be convertible to HA densities using a regression equation.

MicroCT imaging dose to mouse organs using a validated Monte Carlo model of the small animal radiation research platform (SARRP).

The goal of this work was to establish imaging dose to mouse organs with a validated Monte Carlo (MC) model of the image-guided Small Animal Radiation Research Platform (SARRP) and to investigate the effect of scatter from the internal walls on animal therapy dose determination. A MC model of the SARRP was built in the BEAMnrc code and validated with a series of homogeneous and heterogeneous phantom measurements. A segmented microCT scan of a mouse was used in DOSXYZnrc to determine mouse organ microCT imaging doses to 15-35 g mice for the SARRP pancake (mouse lying on couch) and standard (mouse standing on couch) imaging geometries for 40-80 kVp tube voltages. Imaging dose for off-center positioning shifts and maintaining image noise across tube voltages were also calculated. Half-value layer (HVL) measurements for the 220 kVp therapy beam in the presence of the SARRP shielding cabinet were modeled in BEAMnrc and compared to the 100 cm source-to-detector distance (SDD) in the scatter free, narrow-beam geometry recommended by the American Association of Physicists in Medicine Task Group 61 (AAPM TG-61). For a 60 kVp, 0.8 mA, and 60 s scan protocol, maximum mean organ imaging doses to boney and non-boney structures were 10.5 cGy and 3.5 cGy, respectively, for an average size 20 g mouse. Current-exposure combinations above 323, 203, 147, 116, and 95 mAs for 40-80 kVp tube voltages, respectively, will increase body doses above 10 cGy. MicroCT mean body dose was 18% lower in pancake compared to standard imaging geometry. An 11% difference in measured HVL at a 50 cm SDD was found compared to MC simulated HVL for the AAPM TG-61 recommended scatter free geometry at a 100 cm SDD. This change in HVL resulted in a 0.5% change in absorbed dose to water calculations for the treatment beam.

Quantitative assessment of paravalvular leakage after transcatheter aortic valve replacement using a patient-specific pulsatile flow model.

BACKGROUND: Quantitative assessment of post-transcatheter aortic valve replacement (TAVR) aortic regurgitation (AR) remains challenging. We developed patient-specific anatomical models with pulsatile flow circuit and investigated factors associated with AR after TAVR. METHODS: Based on pre-procedural computed tomography (CT) data of the six patients who underwent transfemoral TAVR using a 23-mm SAPIEN XT, anatomically and mechanically equivalent aortic valve models were developed. Forward flow and heart rate of each patient in two days after TAVR were duplicated under mean aortic pressure of 80mmHg. Paravalvular leakage (PVL) volume in basal and additional conditions was measured for each model using an electromagnetic flow sensor. Incompletely apposed tract between the transcatheter and aortic valves was examined using a micro-CT. RESULTS: PVL volume in each patient-specific model was consistent with each patient's PVL grade, and was affected by hemodynamic conditions. PVL and total regurgitation volume increased with the mean aortic pressure, whereas closing volume did not change. In contrast, closing volume increased proportionately with heart rate, but PVL did not change. The minimal cross-sectional gap had a positive correlation with the PVL volumes (r=0.89, P=0.02). The gap areas typically occurred in the vicinity of the bulky calcified nodules under the native commissure. CONCLUSIONS: PVL volume, which could be affected by hemodynamic conditions, was significantly associated with the minimal cross-sectional gap area between the aortic annulus and the stent frame. These data may improve our understanding of the mechanism of the occurrence of post-TAVR PVL.

Reliable landmarks for precise topographical analyses of pathological structural changes of the ovine tibial plateau in 2-D and 3-D subspaces.

Selecting identical topographical locations to analyse pathological structural changes of the osteochondral unit in translational models remains difficult. The specific aim of the study was to provide objectively defined reference points on the ovine tibial plateau based on 2-D sections of micro-CT images useful for reproducible sample harvesting and as standardized landmarks for landmark-based 3-D image registration. We propose 5 reference points, 11 reference lines and 12 subregions that are detectable macroscopically and on 2-D micro-CT sections. Their value was confirmed applying landmark-based rigid and affine 3-D registration methods. Intra- and interobserver comparison showed high reliabilities, and constant positions (standard errors < 1%). Spatial patterns of the thicknesses of the articular cartilage and subchondral bone plate were revealed by measurements in 96 individual points of the tibial plateau. As a case study, pathological phenomena 6 months following OA induction in vivo such as osteophytes and areas of OA development were mapped to the individual subregions. These new reference points and subregions are directly identifiable on tibial plateau specimens or macroscopic images, enabling a precise topographical location of pathological structural changes of the osteochondral unit in both 2-D and 3-D subspaces in a region-appropriate fashion relevant for translational investigations.

Evaluating causes of error in landmark-based data collection using scanners.

In this study, we assess the precision, accuracy, and repeatability of craniodental landmarks (Types I, II, and III, plus curves of semilandmarks) on a single macaque cranium digitally reconstructed with three different surface scanners and a microCT scanner. Nine researchers with varying degrees of osteological and geometric morphometric knowledge landmarked ten iterations of each scan (40 total) to test the effects of scan quality, researcher experience, and landmark type on levels of intra- and interobserver error. Two researchers additionally landmarked ten specimens from seven different macaque species using the same landmark protocol to test the effects of the previously listed variables relative to species-level morphological differences (i.e., observer variance versus real biological variance). Error rates within and among researchers by scan type were calculated to determine whether or not data collected by different individuals or on different digitally rendered crania are consistent enough to be used in a single dataset. Results indicate that scan type does not impact rate of intra- or interobserver error. Interobserver error is far greater than intraobserver error among all individuals, and is similar in variance to that found among different macaque species. Additionally, experience with osteology and morphometrics both positively contribute to precision in multiple landmarking sessions, even where less experienced researchers have been trained in point acquisition. Individual training increases precision (although not necessarily accuracy), and is highly recommended in any situation where multiple researchers will be collecting data for a single project.

Accuracy, precision and inter-rater reliability of micro-CT analysis of false starts on bones. A preliminary validation study.

Micro computed tomography (micro-CT) has already been proposed as a useful technique for the qualitative analysis of false starts (FS) produced on human bones, although the reliability and the error rate of this technique have not been tested yet, neither for qualitative nor for quantitative assessments. The aim of the present study was to test the morphological agreement, accuracy, precision and inter-rater reliability of micro-CT analysis of FS on bones. The morphological agreement was assessed through the degree of concordance among the 3 independent blind raters in the identification of the shape of 24 FS manually produced on bones by 3 different saws (8 FS for each saw). The accuracy was calculated through the percentage of error in the automatic and manual measurement of the diameter of a reference object. The precision was calculated as CV% of multiple measurements performed by 3 independent blind raters on the reference object and one bone sample acquired 20 times. The inter-rater reliability was assessed as intraclass correlation coefficient (ICC) among measurements performed by 3 independent blind raters, assessing 24 FS produced using 3 different saws. The results demonstrated that both qualitative and quantitative analysis were reproducible and robust. Micro-CT analysis showed a 100% morphological agreement, a high level of accuracy (percentage error < 0,5%), precision (CV% < 5%) and inter-rater reliability (ICC > 0.995), when FS were analyzed by forensic pathologists and/or radiologists with adequate expertise. Obviously, further validation studies are needed, including a higher number of samples produced by a wider variety of saws and multiple operators.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Evaluation of Container Closure System Integrity for Frozen Storage Drug Products.

Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Shipment of frozen product could potentially be performed in the frozen state, yet possibly at different temperatures, for example, using dry ice (-80 °C). Container closure systems of drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Below their Tg, rubber stoppers are known to lose their elastic properties and become brittle, and thus potentially fail to maintain container closure integrity in the frozen state. Leaks during frozen temperature storage and transportation are likely to be transient, yet, can possibly risk container closure integrity and lead to microbial contamination. After thawing, the rubber stopper is supposed to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal physical container closure integrity) for direct assessment of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, different container closure systems were evaluated with regard to physical container closure integrity in the frozen state to assess the suitability of vial/stopper combinations and were compared to a gas headspace method. In summary, the thermal physical container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system. LAY ABSTRACT: Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Container closure systems for drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Leaks during frozen temperature storage and transportation are likely to be transient, yet they can possibly risk container closure integrity and lead to microbial contamination and sterility breach. After thawing, the rubber stopper is expected to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal container closure integrity) for direct measurement of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, we found that the thermal container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system.

Avaliação por microtomografia computadorizada da remoção de smear layer e debris em molares inferiores após utilização de diferentes agentes desmineralizantes / Computerized microtomography evaluation of smear layer removal and debris in lower molars after use of different demineralizing agents

O objetivo do estudo foi avaliar, por microtomografia computadorizada, a eficiência da solução de ácido fosfórico 37% e do EDTA 17% na remoção de smear layer e debris de tecidos mineralizados acumulados (DTMA) após o preparo químico-mecânico. Métodos: Vinte e seis raízes mesiais de molares inferiores foram microtomografadas em três momentos distintos: exame inicial, após preparo-químico mecânico e após remoção de smear layer pelo emprego do microtomógrafo Skyscan 1173 utilizando resolução de 14,97 µm. Os canais foram preparados utilizando limas Reciproc R40, NaOCl 6% e o Sistema EndoVac para irrigação/aspiração. Após preparo, os espécimes foram divididos aleatoriamente em dois grupos, de acordo com a solução desmineralizante utilizada: solução de ácido fosfórico 37% ou EDTA 17%. O software CTan foi utilizado para análise volumétrica e o software Seg3D para reconstruções tridimensionais. Os dados foram analisados através do teste Mann-Whitney utilizando α=0.05 como nível de significância estatística. Resultados: A diferença entre os volumes após remoção de smear layer e após o preparo químico-mecânico demonstrou que o ácido fosfórico 37% foi significativamente mais eficiente na remoção de smear layer e DTMA que o EDTA 17% (p<0,05). Conclusões: Demonstrou-se que ambos, ácido fosfórico 37% e EDTA 17%, são agentes eficazes na remoção de smear layer e debris, porém melhores resultados foram obtidos com o uso do ácido fosfórico 37%. (AU)

Introduction: The aim of this study was to evaluate, by micro­computed tomography, the efficiency of 37% phosphoric acid solution with that of 17% EDTA in the removal of smear layer and accumulated hard tissue debris (AHTD) after canal preparation. Methods: Twenty-six mesial roots of mandibular molars were subjected to three microtomographic scannings: initial scanning, after instrumentation and after smear layer removal using the SkyScan 1173 X-ray microtomograph at a resolution of 14,97 µm. The canals were prepared using R40 Reciproc instruments, 6% NaOCl and EndoVac System to irrigation/aspiration. After instrumentation, the specimens were randomly divided into two groups according to the demineralizing solution used: 37% phosphoric acid solution or 17% EDTA. CTAn software was used for volumetric analysis and Seg3D software was used for 3D model reconstructions. Data were analyzed using Mann-Whitney test using α=0.05 as level of statistical significance. Results: The difference between the volumes after removal of smear layer and after chemical-mechanical preparation demonstrated that 37% phosphoric acid was significantly more effective in removing smear layer and AHTD than EDTA 17% (p <0.05). Conclusions: It was shown that both 37% phosphoric acid and 17% EDTA are effective agents in the removal of smear layer and debris, but better results were obtained with the use of 37% phosphoric acid. (AU)

Acurácia de um sistema de diagnóstico de cárie visual e da estereofotografia de superfícies oclusais comparado à um padrão ouro em micro-CT / Acuracy of a carie diagnosis visual system and stereophotography of surface occlusal compared to a gold standard in micro-CT

O objetivo do presente estudo foi verificar a acurácia in vitro do ICDAS visual e baseado em imagens digitais no diagnóstico de cárie oclusal incipiente em molares permanentes e compará-los com um padrão ouro em micro-CT. Para este fim, foram utilizados 40 molares permanentes, obtidos através de um biorrepositório, onde três avaliadores calibrados atribuíram escores ICDAS. Primeiramente os escores foram atribuídos através de exame visual e este foi repetido após uma semana. Em seguida, passado um mês da primeira avaliação visual, os mesmos avaliadores receberam imagens digitais dos espécimes utilizados anteriormente para nova atribuição de scores ICDAS, sendo repetido também após uma semana. Os dentes foram então escaneados através de micro-CT que foi definido como método padrão-ouro para comparação. O coeficiente de correlação de Spearman foi utilizado para comparar as pontuações de consenso com o padrão-ouro. Os métodos de detecção foram comparados por meio de sensibilidade, especificidade, área sob a curva (AUC), valores preditivos positivos e negativos e precisão para dois limiares diferentes (dentes hígidos versus dentes cariados e dentes que necessitam de tratamento operatório versus tratamento não ­ operatório). Os valores médios de kappa para o intra-examinador foram 0,68 e 0,74 para inspeção visual e método baseado em imagens, respectivamente. Os valores de sensibilidade para o limiar de "presença de cárie" no método ICDAS visual e ICDAS fotográfico foram elevados quando comparados com micro-CT (0,93 e 0,97, respectivamente), mesmo com valores de precisão (0,83 e 0,85). Já a especificidade foi mais expressiva para o limiar de "cárie com necessidade de tratamento operatório" com 0,77 e 0,82 para os mesmos métodos. O coeficiente de correlação de Spearman foi de 0,53 e 0,43 (p <0,05). Em vista disso, conclui-se que o ICDAS visual e o baseado em imagens digitais foram semelhantes quanto à exatidão diagnóstica em comparação com um padrão - ouro em micro-CT, podendoe ser uma prosposta para estudos epidemiológicos. (AU)

The aim of the present study was to verify the in vitro accuracy of visual and digital-based ICDAS in the diagnosis of incipient occlusal caries in permanent molars compared to a micro-CT gold standard. For this purpose, 40 permanent molars were obtained from a pool of extracted teeth, cleaned and mounted in gypsum bases where three calibrated examiners assigned ICDAS scores. The scores were attributed by means of visual examination and were repeated after one week. One month after the first visual evaluation, the same examiners received digital images of the previously used specimens for re-assignment of ICDAS scores, which were also repeated after one week. The teeth were then scanned through a micro-CT device which was defined as gold standard method for comparison. The Spearman correlation coefficient was used to compare the consensus scores with the gold standard. The detection methods were compared by means of sensitivity, specificity, area under the curve (AUC), positive and negative predictive values and precision for two different thresholds (healthy teeth X decayed teeth and teeth requiring operative treatment X non - operative treatment). Mean kappa values for the intra-examiner agreement were 0.68 and 0.74 for visual inspection and image-based method, respectively. Sensitivity values for the "decayed teeth" threshold in the visual ICDAS method and the photographic ICDAS were high compared with the micro-CT golden standard (0.93 and 0.97, respectively), even with precision values (0.83 and 0.85). The specificity was more significant for the "caries in need for operative treatment" threshold, with 0.77 and 0.82 for the same methods. The Spearman correlation coefficient was 0.53 and 0.43 (p <0.05). It can be concluded that the visual and digital-based ICDAS were similar in terms of diagnostic accuracy compared to a gold standard in micro-CT, and may be advocated for epidemiological studies. (AU)

Geometric Parameters Estimation and Calibration in Cone-Beam Micro-CT.

The quality of Computed Tomography (CT) images crucially depends on the precise knowledge of the scanner geometry. Therefore, it is necessary to estimate and calibrate the misalignments before image acquisition. In this paper, a Two-Piece-Ball (TPB) phantom is used to estimate a set of parameters that describe the geometry of a cone-beam CT system. Only multiple projections of the TPB phantom at one position are required, which can avoid the rotation errors when acquiring multi-angle projections. Also, a corresponding algorithm is derived. The performance of the method is evaluated through simulation and experimental data. The results demonstrated that the proposed method is valid and easy to implement. Furthermore, the experimental results from the Micro-CT system demonstrate the ability to reduce artifacts and improve image quality through geometric parameter calibration.