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Avoiding artefacts in MicroCT imaging of collagen scaffolds: Effect of phosphotungstic acid (PTA)-staining and crosslink density.

ABSTRACT: X-ray micro-computed tomography (μ-CT) can be used to provide both qualitative and quantitative information on the structure of three-dimensional (3D) bioactive scaffolds. When performed in a dry state, μ-CT accurately reflects the structure of collagen-based scaffolds, but imaging in a wet state offers challenges with radiolucency. Here we have used phosphotungstic acid (PTA) as a contrast agent to visualise fully hydrated collagen scaffolds in a physiologically relevant environment. A systematic investigation was performed to understand the effects of PTA on the results of μ-CT imaging by varying sample processing variables such as crosslinking density, hydration medium and staining duration. Immersing samples in 0.3% PTA solution overnight completely stained the samples and the treatment provided a successful route for μ-CT analysis of crosslinked samples. However, significant structural artefacts were observed for samples which were either non-crosslinked or had low levels of crosslinking, which had a heterogeneous interior architecture with collapsed pores at the scaffold periphery. This work highlights the importance of optimising the choice of processing and staining conditions to ensure accurate visualisation for hydrated 3D collagen scaffolds in an aqueous medium.

SUBMITTER: Kwon KA

PROVIDER: S-EPMC8424391 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Phosphotungstic acid-enhanced microCT: Optimized protocols for embryonic and early postnatal mice.

Project description:BackgroundGiven the need for descriptive and increasingly mechanistic morphological analyses, contrast-enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro-level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA-enhanced microCT.ResultsOur protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution.ConclusionThese protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

| S-EPMC7125040 | biostudies-literature

Optimized data analysis avoiding trypsin artefacts

Project description:Most bottom-up proteomics experiments share two features: The use of trypsin to digest proteins for mass spectrometry and the statistic driven matching of the measured peptide fragment spectra against protein database derived in silico generated spectra. While this extremely powerful approach in combination with latest generation mass spectrometers facilitates very deep proteome coverage, the assumptions made have to be met to generate meaningful results. One of these assumptions is that the measured spectra indeed have a match in the search space, since the search engine will always report the best match. However, one of the most abundant proteins in the sample, the protease, is often not represented in the employed database. It is therefore widely accepted in the community to include the protease and other common contaminants in the database to avoid false positive matches. Although this approach accounts for unmodified trypsin peptides, the most widely employed trypsin preparations are chemically modified to prevent autolysis and premature activity loss of the protease. In this study we observed numerous spectra of modified trypsin derived peptides in samples from our laboratory as well as in datasets downloaded from public repositories. In many cases the spectra were assigned to other proteins, often with good statistical significance. We therefore designed a new database search strategy employing an artificial amino acid which accounts for these peptides with a minimal increase in search space and the concomitant loss of statistical significance. Moreover, this approach can be easily implemented into existing workflows for many widely used search engines.

2016-04-15 | PXD002726 | Pride

Reducing soft-tissue shrinkage artefacts caused by staining with Lugol's solution.

Project description:Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is progressively used in clinical and morphological research to study developmental anatomy. Lugol's solution (Lugol) has gained interest as an effective contrast agent; however, usage is limited due to extensive soft-tissue shrinkage. The mechanism of Lugol-induced shrinkage and how to prevent it is largely unknown, hampering applications of Lugol in clinical or forensic cases where tissue shrinkage can lead to erroneous diagnostic conclusions. Shrinkage was suggested to be due to an osmotic imbalance between tissue and solution. Pilot experiments pointed to acidification of Lugol, but the relation of acidification and tissue shrinkage was not evaluated. In this study, we analyzed the relation between tissue shrinkage, osmolarity and acidification of the solution during staining. Changes in tissue volume were measured on 2D-segmented magnetic resonance and diceCT images using AMIRA software. Partial correlation and stepwise regression analysis showed that acidification of Lugol is the main cause of tissue shrinkage. To prevent acidification, we developed a buffered Lugol's solution (B-Lugol) and showed that stabilizing its pH almost completely prevented shrinkage without affecting staining. Changing from Lugol to B-Lugol is a major improvement for clinical and morphological research and only requires a minor adaptation of the staining protocol.

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Influence of Crosslink Density and Stiffness on Mechanical Properties of Type I Collagen Gel.

Project description:The mechanical properties of type I collagen gel vary due to different polymerization parameters. In this work, the role of crosslinks in terms of density and stiffness on the macroscopic behavior of collagen gel were investigated through computational modeling. The collagen fiber network was developed in a representative volume element, which used the inter-fiber spacing to regulate the crosslink density. The obtained tensile behavior of collagen gel was validated against published experimental data. Results suggest that the cross-linked fiber alignment dominated the strain stiffening effect of the collagen gel. In addition, the gel stiffness was enhanced approximately 40 times as the crosslink density doubled. The non-affine deformation was reduced with the increased crosslink density. A positive bilinear correlation between the crosslink density and gel stiffness was obtained. On the other hand, the crosslink stiffness had much less impact on the gel stiffness. This work could enhance our understanding of collagen gel mechanics and shed lights on designing future clinical relevant biomaterials with better control of polymerization parameters.

| S-EPMC5455287 | biostudies-other

Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

Project description:When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ?10??M. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ?190,000 actin molecules (?500??m) in the fission yeast contractile ring. These filaments shorten from ?500 to ?100 subunits as the ring constricts.

| S-EPMC5509211 | biostudies-literature

Collagen crosslink excretion and staging of oral cancer.

Project description:Lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) are collagen crosslink residues of which the urinary concentration reflects the level of connective-tissue turnover. HP is ubiquitous in tissue, whereas LP is specific for bone. The purpose of this investigation was to assess the sensitivity and specificity of an increased urinary concentration of both HP and LP in indicating infiltration of mandibular bone by an oral squamous cell carcinoma (OSCC) or recurrence of the disease after successful therapy. We investigated the history and urine levels in 116 adult patients, who were divided into the following groups. Group 1: patients with OSCC with bone infiltration (n=17); group 2: patients with confirmed OSCC (n=12) without evidence of bone infiltration; group 3: patients with recurrence of an OSCC (n=13); group 4: patients without clinical evidence of disease (n=74). The range and upper limit of normal values (HP(max) and LP(max)) were measured from the normal controls in group 4. Levels of LP and HP were measured by HPLC and fluorescence detection. There was a significant difference in the average urinary levels of LP and HP between groups 1-4 (P<0.001). The presence of mandibular bone infiltration could be detected with a sensitivity and specificity of 100% when comparing groups 1 and 2. Presence of tumour tissue could be detected with a sensitivity of 90%. In conclusion, a normal LP concentration in patients with an OSCC strongly suggests that bone invasion by the disease has not taken place. If both urinary HP and LP are elevated, disease recurrence is highly likely.

| S-EPMC2376365 | biostudies-other

Tunable tissue scaffolds fabricated by in situ crosslink in phase separation system.

Project description:Three-dimensional (3-D) scaffolds with intrinsic porous structures are desirable in various tissue regeneration applications. In this study, a unique method that combines thermally induced phase separation with a photocrosslinking process was developed for the fabrication of 3-D crosslinked polymer scaffolds with densely interconnected porous structures. Biodegradable poly(propylene fumarate)-co-poly(L-lactic acid) with crosslinkable fumarate bonds were used as the structural polymer material and a dioxane/water binary system was applied for the phase separation. By altering the polymer composition (9, 5 and 3 wt%), different types of scaffolds with distinct morphology, mechanical strength, degradation rate, cell growth and morphology, and extracellular matrix production were fabricated. These crosslinked 3-D porous scaffolds with tunable strength and biological responses show promise for potential applications in regenerative therapies, including bone and neural tissue engineering.

| S-EPMC4792294 | biostudies-other

3D collagen-based scaffolds for biophysical tumour microenvironment studies

Project description:3D scaffolds collagen I-based were crosslinked with different percentages of 1, 4-butanediol diglycidyl ether (BDDGE) to mimic native tissue and tumour tissue. Normal fibroblasts (NFs) or cancer-associated fibroblasts (CAFs) were added to the system to assess how mechanical features influence stromal compartment in native or tumour-like systems.

2022-01-12 | GSE168639 | GEO

MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.

Project description:BackgroundComparative, functional, and developmental studies of animal morphology require accurate visualization of three-dimensional structures, but few widely applicable methods exist for non-destructive whole-volume imaging of animal tissues. Quantitative studies in particular require accurately aligned and calibrated volume images of animal structures. X-ray microtomography (microCT) has the potential to produce quantitative 3D images of small biological samples, but its widespread use for non-mineralized tissues has been limited by the low x-ray contrast of soft tissues. Although osmium staining and a few other techniques have been used for contrast enhancement, generally useful methods for microCT imaging for comparative morphology are still lacking.ResultsSeveral very simple and versatile staining methods are presented for microCT imaging of animal soft tissues, along with advice on tissue fixation and sample preparation. The stains, based on inorganic iodine and phosphotungstic acid, are easier to handle and much less toxic than osmium, and they produce high-contrast x-ray images of a wide variety of soft tissues. The breadth of possible applications is illustrated with a few microCT images of model and non-model animals, including volume and section images of vertebrates, embryos, insects, and other invertebrates. Each image dataset contains x-ray absorbance values for every point in the imaged volume, and objects as small as individual muscle fibers and single blood cells can be resolved in their original locations and orientations within the sample.ConclusionWith very simple contrast staining, microCT imaging can produce quantitative, high-resolution, high-contrast volume images of animal soft tissues, without destroying the specimens and with possibilities of combining with other preparation and imaging methods. Such images are expected to be useful in comparative, developmental, functional, and quantitative studies of morphology.

| S-EPMC2717911 | biostudies-literature

Standard method for microCT-based additive manufacturing quality control 2: Density measurement.

Project description:MicroCT is best known for its ability to detect and quantify porosity or defects, and to visualize its 3D distribution. However, it is also possible to obtain accurate volumetric measurements from parts - this can be used in combination with the part mass to provide a good measure of its average density. The advantage of this density-measurement method is the ability to combine the density measurement with visualization and other microCT analyses of the same sample. These other analyses may include detailed porosity or void analysis (size and distribution) and roughness assessment, obtainable with the same scan data. Simple imaging of the interior of the sample allows the detection of unconsolidated powder, open porosity to the surface or the presence of inclusions. The CT density method presented here makes use of a 10 mm cube sample and a simple data analysis workflow, facilitating standardization of the method. A laboratory microCT scanner is required at 15 μm voxel size, suitable software to allow sub-voxel precise edge determination of the scanned sample and hence an accurate total volume measurement, and a scale with accuracy to 3 digits. •MicroCT-based mean density measurement method.•Accurate volume measurement and scale mass.•10 mm cube sample allows standardization and automation of workflow.

| S-EPMC6168933 | biostudies-literature

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