Contrast-Enhanced Micro-CT Imaging of Murine Mandibles: A Multi-Method Approach for Simultaneous Hard and Soft Tissue Analysis.

AIM: To develop and evaluate a novel multi-method micro-computed tomography (µCT) imaging protocol for enhanced visualization of both hard and soft tissues in murine mandibles, addressing the limitations of traditional imaging techniques in dental research. MATERIALS AND METHODS: We employed a contrast-enhanced (CE) µCT imaging technique using Lugol's iodine as a contrast agent to visualize the intricate structures of murine mandibles. The protocol involved the combination of conventional µCT imaging as well as CE-µCT, including decalcification with EDTA, allowing for simultaneous assessment of hard and soft tissues. The method is compared with standard imaging modalities, and the ability to visualize detailed anatomical features is discussed. RESULTS: The CE-µCT imaging technique provided superior visualization of murine mandibular structures, including dental pulp, periodontal ligaments and the surrounding soft tissues, along with conventional µCT imaging of alveolar bone and teeth. This method revealed detailed anatomical features with high specificity and contrast, surpassing traditional imaging approaches. CONCLUSION: Our findings demonstrate the potential of CE-µCT imaging with Lugol's iodine as a powerful tool for dental research. This technique offers a comprehensive view of the murine mandible, facilitating advanced studies in tissue engineering, dental pathology and the development of dental materials.

Morphometric micro-CT study of contralateral mandibular incisors.

OBJECTIVES: This study aimed to determine the degree of similarity and symmetry in the anatomy of contralateral mandibular incisors. Three-dimensional (3D) models of extracted teeth were obtained from microtomography (micro-CT) scans. Qualitative and quantitative assessments of the morphology and comparison of contralateral pairs were made. The null hypothesis was that contralateral mandibular incisors could not be considered identical in simple morphometric measurements. METHODS: Sixty pairs of mandibular incisors were extracted from 30 patients and scanned with micro-CT. Virtual models of the cemento-enamel junction to the root apex were rendered. Parameters such as length, canal width, dentinal thicknesses, tortuosity, centerline length, accessory canals, root canal configurations, and root canal orifice cross-sections were used to compare the teeth. Width and thickness comparisons between paired teeth in the same individual were made by paired t-test (Wilcoxon signed-rank test for variables not normally distributed). An online randomization tool generated randomized pairs (independent of the individual/patient). Subsequently, an unpaired t-test (or Mann-Whitney U test for non-normally distributed parameters) and a correlation analysis were conducted. Canal configurations were classified according to preexisting classification schemes. The number and location of accessory canals and apical foramina were registered and compared. RESULTS: Utilizing advanced imaging techniques and quantitative analyses, our study establishes that contralateral mandibular incisors exhibit a remarkable degree of symmetry in multiple morphological parameters, including length, canal width, and dentinal thicknesses. The apical third showed a high degree of inter-variability for the contralateral pairs. The rigorous statistical analysis of the normalized parameters by Z-score showed no statistically significant differences between the contralateral mandibular incisors. Comparisons between central and lateral teeth revealed differences in root length but no significant disparity in the distribution of accessory canals. Central teeth, on average, were longer, while accessory canals were distributed relatively evenly between central and lateral teeth. CONCLUSIONS: The findings of this study further establish the significant similarities between contralateral mandibular incisors, reinforcing their suitability as a reliable substrate for root canal comparison studies. CLINICAL RELEVANCE: The absence of statistically significant differences between contralateral pairs in normalized parameters underscores their potential as a reliable reference point for root canal comparison studies in clinical dentistry. Furthermore, our findings emphasize the importance of individualized treatment planning, considering the natural symmetry in mandibular incisors to enhance clinical decision-making. This research contributes valuable insights to the field of endodontics, offering a standardized approach to sample selection and enriching the understanding of dental anatomy.

Irisin reduces orthodontic tooth movement in rats by promoting the osteogenic potential in the periodontal ligament.

OBJECTIVES: Positive effects of irisin on osteogenic differentiation of periodontal ligament (PDL) cells have been identified previously, this study aims to examine its effect on orthodontic tooth movement (OTM) in vivo. MATERIALS AND METHODS: The maxillary right first molars of male Wistar rats (n = 21) were moved mesially for 14 days, with submucosal injection of two dosages of irisin (0.1 or 1 µg) or phosphate-buffered saline (control) every third day. OTM was recorded by feeler gauge and micro-computed tomography (µCT). Alveolar bone and root volume were analysed using µCT, and plasma irisin levels by ELISA. Histological characteristics of PDL tissues were examined, and the expression of collagen type I, periostin, osteocalcin (OCN), von Willebrand factor (vWF) and fibronectin type III domain-containing protein 5 (FNDC5) in PDL was evaluated by immunofluorescence staining. RESULTS: Repeated 1 µg irisin injections suppressed OTM on days 6, 9, and 12. No significant differences were observed in OTM in the 0.1 µg irisin group, or in bone morphometric parameters, root volume or plasma irisin, compared to control. Resorption lacunae and hyalinization were found at the PDL-bone interface on the compression side in the control, whereas they were scarce after irisin administration. The expression of collagen type I, periostin, OCN, vWF, and FNDC5 in PDL was enhanced by irisin administration. LIMITATIONS: The feeler gauge method may overestimate OTM. CONCLUSIONS: Submucosal irisin injection reduced OTM by enhancing osteogenic potential of PDL, and this effect was more significant on the compression side.

Influence of Drug Load on the Printability and Solid-State Properties of 3D-Printed Naproxen-Based Amorphous Solid Dispersion.

Fused deposition modelling-based 3D printing of pharmaceutical products is facing challenges like brittleness and printability of the drug-loaded hot-melt extruded filament feedstock and stabilization of the solid-state form of the drug in the final product. The aim of this study was to investigate the influence of the drug load on printability and physical stability. The poor glass former naproxen (NAP) was hot-melt extruded with Kollidon® VA 64 at 10-30% w/w drug load. The extrudates (filaments) were characterised using differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and thermogravimetric analysis (TGA). It was confirmed that an amorphous solid dispersion was formed. A temperature profile was developed based on the results from TGA, DSC, and DMA and temperatures used for 3D printing were selected from the profile. The 3D-printed tablets were characterised using DSC, X-ray computer microtomography (XµCT), and X-ray powder diffraction (XRPD). From the DSC and XRPD analysis, it was found that the drug in the 3D-printed tablets (20 and 30% NAP) was amorphous and remained amorphous after 23 weeks of storage (room temperature (RT), 37% relative humidity (RH)). This shows that adjusting the drug ratio can modulate the brittleness and improve printability without compromising the physical stability of the amorphous solid dispersion.

Correction to: the Effect of Cross-Linking Efficiency of Drug-Loaded Novel Freeze Gelated Chitosan Templates for Periodontal Tissue Regeneration.

The correct spelling of the second author's name is Liebert Parreiras Nogueira.

Impact of drug load and polymer molecular weight on the 3D microstructure of printed tablets.

This study investigates the influence of drug load and polymer molecular weight on the structure of tablets three-dimensionally (3D) printed from the binary mixture of prednisolone and hydroxypropyl methylcellulose (HPMC). Three different HPMC grades, (AFFINISOLTM HPMC HME 15LV, 90 Da (HPMC 15LV); 100LV, 180 Da (HPMC 100LV); 4M, 500 Da (HPMC 4M)), which are suitable for hot-melt extrusion (HME), were used in this study. HME was used to fabricate feedstock material, i.e., filaments, at the lowest possible extrusion temperature. Filaments of the three HPMC grades were prepared to contain 2.5, 5, 10 and 20 % (w/w) prednisolone. The thermal degradation of the filaments was studied with thermogravimetric analysis, while solid-state properties of the drug-loaded filaments were assessed with the use of X-ray powder diffraction. Prednisolone in the freshly extruded filaments was determined to be amorphous for drug loads up to 10%. It remained physically stable for at least 6 months of storage, except for the filament containing 10% drug with HPMC 15LV, where recrystallization of prednisolone was detected. Fused deposition modeling was utilized to print honeycomb-shaped tablets from the HME filaments of HPMC 15LV and 100LV. The structural characteristics of the tablets were evaluated using X-ray microcomputed tomography, specifically porosity and size of structural elements were investigated. The tablets printed from HPMC 15LV possessed in general lower total porosity and pores of smaller size than tablets printed from the HPMC 100LV. The studied drug loads were shown to have minor effect on the total porosity of the tablets, though the lower the drug load was, the higher the variance of porosity along the height of the tablet was observed. It was found that tablets printed with HPMC 15LV showed higher structural similarity with the virtually designed model than tablets printed from HPMC 100LV. These findings highlight the relevance of the drug load and polymer molecular weight on the microstructure and structural properties of 3D printed tablets.

Contrast-enhanced Micro-CT 3D visualization of cell distribution in hydrated human cornea.

Background: The cornea, a vital component of the human eye, plays a crucial role in maintaining visual clarity. Understanding its ultrastructural organization and cell distribution is fundamental for elucidating corneal physiology and pathology. This study comprehensively examines the microarchitecture of the hydrated human cornea using contrast-enhanced micro-computed tomography (micro-CT). Method: Fresh human corneal specimens were carefully prepared and hydrated to mimic their in vivo state. Contrast enhancement with Lugol's iodine-enabled high-resolution Micro-CT imaging. The cells' three-dimensional (3D) distribution within the cornea was reconstructed and analyzed. Results: The micro-CT imaging revealed exquisite details of the corneal ultrastructure, including the spatial arrangement of cells throughout its depth. This novel approach allowed for the visualization of cells' density and distribution in different corneal layers. Notably, our findings highlighted variations in cell distribution between non-hydrated and hydrated corneas. Conclusions: This study demonstrates the potential of contrast-enhanced micro-CT as a valuable tool for non-destructive, 3D visualization and quantitative analysis of cell distribution in hydrated human corneas. These insights contribute to a better understanding of corneal physiology and may have implications for research in corneal diseases and tissue engineering.

Enhanced Bone Healing in Critical-Sized Rabbit Femoral Defects: Impact of Helical and Alternate Scaffold Architectures.

This study investigates the effect of scaffold architecture on bone regeneration, focusing on 3D-printed polylactic acid-bioceramic calcium phosphate (PLA-bioCaP) composite scaffolds in rabbit femoral condyle critical defects. We explored two distinct scaffold designs to assess their influence on bone healing and scaffold performance. Structures with alternate (0°/90°) and helical (0°/45°/90°/135°/180°) laydown patterns were manufactured with a 3D printer using a fused deposition modeling technique. The scaffolds were meticulously characterized for pore size, strut thickness, porosity, pore accessibility, and mechanical properties. The in vivo efficacy of these scaffolds was evaluated using a femoral condyle critical defect model in eight skeletally mature New Zealand White rabbits. Then, the results were analyzed micro-tomographically, histologically, and histomorphometrically. Our findings indicate that both scaffold architectures are biocompatible and support bone formation. The helical scaffolds, characterized by larger pore sizes and higher porosity, demonstrated significantly greater bone regeneration than the alternate structures. However, their lower mechanical strength presented limitations for use in load-bearing sites.

On the Sorbent Ability and Reusability of Graphene-Oxide-Chitosan Aerogels for the Removal of Dyes from Wastewater.

One of the most persistent issues affecting people worldwide is water contamination due to the indiscriminate disposal of pollutants, causing severe environmental problems. Dyes are among the most harmful contaminants because of their high chemical stability and consequently difficult degradation. To remove contaminants from water, adsorption is the most widely used and effective method. In this work, we recall the results already published about the synthesis, the characterization and the use of porous graphene-oxide-chitosan aerogels as a sorbent material. Those systems, prepared by mixing GO sheets and CS chains, using APS as a cross-linking agent, and by further lyophilization, were further characterized using nano-computed tomography, supplying more understanding about their micro and nano-structure. Their sorbent ability has been investigated also by the study of their isotherm of adsorption of two different anionic dyes: Indigo Carmine and Cibacron Brilliant Yellow. Those analyses confirmed the potentialities of the aerogels and their affinity for those anionic dyes. Moreover, the possibility of regenerating and reusing the material was evaluated as a key aspect for applications of this kind. The treatment with NaOH, to promote the desorption of adsorbed dyes, and subsequent washing with HCl, to re-protonate the system, ensured the regeneration of the gels and their use in multiple cycles of adsorption with the selected water contaminants.

Durability assessment of hydrogel mountings for contrast-enhanced micro-CT.

Micro-computed tomography (micro-CT) provides valuable data for studying soft tissue, though it is often affected by sample movement during scans and low contrast in X-ray absorption. This can result in lower image quality and geometric inaccuracies, collectively known as 'artefacts'. To mitigate these issues, samples can be embedded in hydrogels and enriched with heavy metals for contrast enhancement. However, the long-term durability of these enhancements remains largely unexplored. In this study, we examine the effects of two contrast enhancement agents - iodine and phosphotungstic acid (PTA) - and two hydrogels - agarose and Poloxamer 407 - over a 14-day period. We used Drosophila melanogaster as a test model for our investigation. Our findings reveal that PTA and agarose are highly durable, while iodine and poloxamer hydrogel exhibits higher leakage rates. These observations lay the foundation for estimating contrast stabilities in contrast-enhanced micro-CT with hydrogel embedding and serve to inform future research in this field.

Assessment of the Root Canal Similarity in Contralateral Mandibular Incisors.

INTRODUCTION: The purpose of this study was to determine the degree of similarity between contralateral mandibular incisors utilising 3-dimensional (3D) models obtained from micro-computed tomographic (micro-CT) scans of extracted human teeth. The null hypothesis was that contralateral mandibular incisors do not exhibit matching symmetry. METHODS: Sixty pairs (n = 120) of extracted mandibular incisors were obtained from 30 patients and scanned with micro-CT with a voxel size of 15.0 µm. 3D virtual models of the pulpal cavities were rendered. Geometric morphometric deviation analysis was performed after mirroring, automatic alignment, and co-registration of the models of contralateral teeth root mean square (RMS) errors were calculated. The quantitative analysis of the 3D models included 6 different geometric parameters. Data sets were examined with a 2-sample Kolmogorov-Smirnov test. Post hoc retrospective power analysis was performed to find statistical power (α = 0.05). RESULTS: Contralateral pairs had a narrower distribution in deviation than random pairs. Also, contralateral pairs showed a statistically higher similarity coefficient (5 out of 6 geometric parameters) compared to random pairs (P < .001); no difference was found when comparing central to lateral pairs or between Vertucci type I configurations compared to non-type I. RMS errors had significantly lower Contralateral premolars (CPs) values than random pairs (P < .001). CONCLUSIONS: A high degree of similarity was demonstrated for pairing contralateral mandibular incisors using 3D models. The similarity between contralateral central and lateral incisors suggests that when screened and matched, these 4 teeth might be used in endodontic research where similar root canal anatomy is crucial.

Photoannealing of Microtissues Creates High-Density Capillary Network Containing Living Matter in a Volumetric-Independent Manner.

The vascular tree is crucial for the survival and function of large living tissues. Despite breakthroughs in 3D bioprinting to endow engineered tissues with large blood vessels, there is currently no approach to engineer high-density capillary networks into living tissues in a scalable manner. Here, photoannealing of living microtissue (PALM) is presented as a scalable strategy to engineer capillary-rich tissues. Specifically, in-air microfluidics is used to produce living microtissues composed of cell-laden microgels in ultrahigh throughput, which can be photoannealed into a monolithic living matter. Annealed microtissues inherently give rise to an open and interconnected pore network within the resulting living matter. Interestingly, utilizing soft microgels enables microgel deformation, which leads to the uniform formation of capillary-sized pores. Importantly, the ultrahigh throughput nature underlying the microtissue formation uniquely facilitates scalable production of living tissues of clinically relevant sizes (>1 cm3 ) with an integrated high-density capillary network. In short, PALM generates monolithic, microporous, modular tissues that meet the previously unsolved need for large engineered tissues containing high-density vascular networks, which is anticipated to advance the fields of engineered organs, regenerative medicine, and drug screening.

Adipose-Derived Stromal Cells Preserve Pancreatic Islet Function in a Transplantable 3D Bioprinted Scaffold.

Intra-portal islet transplantation is currently the only clinically approved beta cell replacement therapy, but its outcome is hindered by limited cell survival due to a multifactorial reaction against the allogeneic tissue in liver. Adipose-derived stromal cells (ASCs) can potentially improve the islet micro-environment by their immunomodulatory action. The challenge is to combine both islets and ASCs in a relatively easy and consistent long-term manner in a deliverable scaffold. Manufacturing the 3D bioprinted double-layered scaffolds with primary islets and ASCs using a mix of alginate/nanofibrillated cellulose (NFC) bioink is reported. The diffusion properties of the bioink and the supportive effect of human ASCs on islet viability, glucose sensing, insulin secretion, and reducing the secretion of pro-inflammatory cytokines are demonstrated. Diabetic mice transplanted with islet-ASC scaffolds reach normoglycemia seven days post-transplantation with no significant difference between this group and the group received islets under the kidney capsules. In addition, animals transplanted with islet-ASC scaffolds stay normoglycemic and show elevated levels of C-peptide compared to mice transplanted with islet-only scaffolds. The data present a functional 3D bioprinted scaffold for islets and ASCs transplanted to the extrahepatic site and suggest a possible role of ASCs on improving the islet micro-environment.

Void Content Determination of Carbon Fiber Reinforced Polymers: A Comparison between Destructive and Non-Destructive Methods.

The properties of composite materials are highly dependent on the fiber and matrix fraction and on the porosity resulting from micro voids. This paper addresses void content characterization and the constituent content of composite materials by resorting to a comparison of destructive and non-destructive methods. The work presents the detailed procedures of two destructive methods, using acid digestion of epoxy resins matrices, and compares their processes. It also presents the results of a non-destructive method, by means of Micro Computed Tomography (MicroCT). The results of both destructive and non-destructive methods are compared, and a recommendation is made based on the application and the type of composite being analyzed. The MicroCT showed better and more consistent results in detecting voids in the material, while the acid digestion tests provided better results about the fiber and matrix percentage. Exported results from the MicroCT scanning with actual locations of voids were used in numerical analysis, to examine the feasibility of using them, whether by developing models that map damage in the proximity of the void, or by developing models that predict the properties of the entire material with respect to the content, shape, and distribution in the material.

Assessing Anatomical Changes in Male Reproductive Organs in Response to Larval Crowding Using Micro-computed Tomography Imaging.

Ecological conditions shape (adaptive) responses at the molecular, anatomical, and behavioral levels. Understanding these responses is key to predict the outcomes of intra- and inter-specific competitions and the evolutionary trajectory of populations. Recent technological advances have enabled large-scale molecular (e.g., RNAseq) and behavioral (e.g., computer vision) studies, but the study of anatomical responses to ecological conditions has lagged behind. Here, we highlight the role of X-ray micro-computed tomography (micro-CT) in generating in vivo and ex vivo 3D imaging of anatomical structures, which can enable insights into adaptive anatomical responses to ecological environments. To demonstrate the application of this method, we manipulated the larval density of Drosophila melanogaster Meigen flies and applied micro-CT to investigate the anatomical responses of the male reproductive organs to varying intraspecific competition levels during development. Our data is suggestive of two classes of anatomical responses which broadly agree with sexual selection theory: increasing larval density led to testes and ejaculatory duct to be overall larger (in volume), while the volume of accessory glands and, to a lesser extent, ejaculatory duct decreased. These two distinct classes of anatomical responses might reflect shared developmental regulation of the structures of the male reproductive system. Overall, we show that micro-CT can be an important tool to advance the study of anatomical (adaptive) responses to ecological environments.

Synchrotron X-ray biosample imaging: opportunities and challenges.

Synchrotron radiation phase-contrast microtomography is sensitive to low attenuating tissues, giving an alternative visualisation of the sample and being useful for investigating microstructure inside biological specimens without staining them with a contrast medium. The phase-contrast technique has been widely used in the scientific community, as it is a technique associated with radiography and microscopy and able to enhance contrast in soft tissues, specifically at the edges, showing details that could not be seen by the absorption technique. This work aims to show the ability of synchrotron-based phase-contrast microtomography for the visualisation of soft tissues and hard internal structures of millimetre-sized biological organisms. Case studies of the anatomy of Rhodnius prolixus head and Thoropa miliaris tadpole are presented to illustrate the imaging technique.

Dual-functional porous and cisplatin-loaded polymethylmethacrylate cement for reconstruction of load-bearing bone defect kills bone tumor cells.

Malignant bone tumors are usually treated by resection of tumor tissue followed by filling of the bone defect with bone graft substitutes. Polymethylmethacrylate (PMMA) cement is the most commonly used bone substitute in clinical orthopedics in view of its reliability. However, the dense nature of PMMA renders this biomaterial unsuitable for local delivery of chemotherapeutic drugs to limit the recurrence of bone tumors. Here, we introduce porosity into PMMA cement by adding carboxymethylcellulose (CMC) to facilitate such local delivery of chemotherapeutic drugs, while retaining sufficient mechanical properties for bone reconstruction in load-bearing sites. Our results show that the mechanical strength of PMMA-based cements gradually decreases with increasing CMC content. Upon incorporation of ≥3% CMC, the PMMA-based cements released up to 18% of the loaded cisplatin, in contrast to cements containing lower amounts of CMC which only released less than 2% of the cisplatin over 28 days. This release of cisplatin efficiently killed osteosarcoma cells in vitro and the fraction of dead cells increased to 91.3% at day 7, which confirms the retained chemotherapeutic activity of released cisplatin from these PMMA-based cements. Additionally, tibias filled with PMMA-based cements containing up to 3% of CMC exhibit comparable compressive strengths as compared to intact tibias. In conclusion, we demonstrate that PMMA cements can be rendered therapeutically active by introducing porosity using CMC to allow for release of cisplatin without compromising mechanical properties beyond critical levels. As such, these data suggest that our dual-functional PMMA-based cements represent a viable treatment option for filling bone defects after bone tumor resection in load-bearing sites.

Using Copper-Doped Mesoporous Bioactive Glass Nanospheres to Impart Anti-Bacterial Properties to Dental Composites.

Experimental dental resin composites containing copper-doped mesoporous bioactive glass nanospheres (Cu-MBGN) were developed to impart anti-bacterial properties. Increasing amounts of Cu-MBGN (0, 1, 5 and 10 wt%) were added to the BisGMA/TEGDMA resin matrix containing micro- and nano-fillers of inert glass, keeping the resin/filler ratio constant. Surface micromorphology and elemental analysis were performed to evaluate the homogeneous distribution of filler particles. The study investigated the effects of Cu-MBGN on the degree of conversion, polymerization shrinkage, porosity, ion release and anti-bacterial activity on S. mutans and A. naeslundii. Experimental materials containing Cu-MBGN showed a dose-dependent Cu release with an initial burst and a further increase after 28 days. The composite containing 10% Cu-MBGN had the best anti-bacterial effect on S. mutans, as evidenced by the lowest adherence of free-floating bacteria and biofilm formation. In contrast, the 45S5-containing materials had the highest S. mutans adherence. Ca release was highest in the bioactive control containing 15% 45S5, which correlated with the highest number of open porosities on the surface. Polymerization shrinkage was similar for all tested materials, ranging from 3.8 to 4.2%, while the degree of conversion was lower for Cu-MBGN materials. Cu-MBGN composites showed better anti-bacterial properties than composites with 45S5 BG.

Nano-CT as tool for characterization of dental resin composites.

Technological advances have made it possible to examine dental resin composites using 3D nanometer resolution. This investigation aims to characterize existing dental nano-hybrid and micro-hybrid resin composites through comparing and contrasting nano-computed tomography (nano-CT) with micro-CT and high-resolution SEM images. Eight commercially available and widely used dental resin composites, 2 micro-hybrid and 6 nano-hybrid were researched. Cured samples were examined and characterized using nano-CT (resolution 450 nm) and compared with micro-CT images (resolution 2 µm). Acquired images were reconstructed and image analysis was carried out to determine porosity and pore morphology. A comprehensive comparison of scanning micrograph images unsurprisingly revealed that the nano-CT images displayed greater detail of the ultrastructure of cured dental resin composites. Filler particle diameters and its volumes were lower when measured using nano-CT, porosity being higher where analysed at higher resolution. There were large variations between the examined materials. Fewer voids were found in Tetric EvoCeram and IPS Empress Direct, the smallest pores being found in Universal XTE and Tetric EvoCeram. Nano-CT was successfully used to investigate the morphology of dental resin composites and showed that micro-CT gives a lower porosity and pore size but overestimates filler particle size. There were large discrepancies between the tested composites. Evidence of porosities and pores within a specimen is a critical finding and it might have a detrimental effect on a material's clinical performance.