This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2018-0099). The authors thank the people who very generously donated their bodies to Yonsei University College of Medicine. We are grateful to Jun Ho Kim and Jong Ho Bang for their technical support (staff members in the Surgical Anatomy Education Centre at the Yonsei University College of Medicine). We are also grateful to Genoss Co., Ltd. for the high-quality microCT scanning system used in this research.

All cadavers utilized in this study were legally donated to the Surgical Anatomy Education Centre at Yonsei University College of Medicine.
1. Obtaining samples
<ol>
	<li>Draw an incision line on the cadaver with a colored pencil to indicate the cutting area for sample harvesting. Check that the incision line drawn extends medially to a medial canthus, laterally to a lateral canthus, superiorly to a superior border of the lower eyelid, and inferiorly to 1 cm below the line from the orbital r...

<ol>
	<li>Nierenberger, M., Remond, Y., Ahzi, S., Choquet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+three-dimensional+collagen+network+in+soft+tissues+using+contrast+agents+and+high+resolution+micro-CT:+Application+to+porcine+iliac+veins.">Assessing the three-dimensional collagen network in soft tissues using contrast agents and high resolution micro-CT: Application to porcine iliac veins.</a> Comptes Rendus Biologies. 338 (7), 425-433 (2015).</li><li>Vymazalov&#225;, K., Vargov&#225;, L., Zikmund, T., Kaiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+possibilities+of+studying+human+embryos+and+foetuses+using+micro-CT:+a+technical+note.">The possibilities of studying human embryos and foetuses using micro-CT: a technical note.</a> Anatomical Science International. 92 (2), 299-303 (2017).</li><li>Tesa&#345;ov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+micro+computed-tomography+and+3D+printing+for+reverse+engineering+of+mouse+embryo+nasal+capsule.">Use of micro computed-tomography and 3D printing for reverse engineering of mouse embryo nasal capsule.</a> Journal of Instrumentation. 11 (3), 1-11 (2016).</li><li>Nemetschek, T., Riedl, H., Jonak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topochemistry+of+the+binding+of+phosphotungstic+acid+to+collagen.">Topochemistry of the binding of phosphotungstic acid to collagen.</a> Journal of Molecular Biology. 133 (1), 67-83 (1979).</li><li>Rao, R. N., Fallman, P. M., Falls, D. G., Meloan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+PAS-phosphotungstic+acid-Diamine+Supra+Blue+FGL+and+immunological+reactions+for+type+I+collagen.">A comparative study of PAS-phosphotungstic acid-Diamine Supra Blue FGL and immunological reactions for type I collagen.</a> Histochemistry. 91 (4), 283-289 (1989).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+comparative+morphology:+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues.">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiology. 9 (11), (2009).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+Developmental+Biology:+A+Versatile+Tool+for+High-Contrast+3D+Imaging+at+Histological+Resolutions.">MicroCT for Developmental Biology: A Versatile Tool for High-Contrast 3D Imaging at Histological Resolutions.</a> Developmental Dynamics. 238 (3), 632-640 (2009).</li><li>Nieminen, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+collagen+distribution+in+articular+cartilage+using+contrastenhanced+micro-computed+tomography.">Determining collagen distribution in articular cartilage using contrastenhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 23 (9), 1613-1621 (2015).</li><li>Kwon, O. J., Kwon, H., Choi, Y., Cho, T., Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+the+orbicularis+retaining+ligament:+an+anatomical+study+using+micro+computed+tomography.">Three-dimensional structure of the orbicularis retaining ligament: an anatomical study using micro computed tomography.</a> Scientific Reports. 8 (1), 17042 (2018).</li><li>Dullin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#956;CT+of+ex-vivo+stained+mouse+hearts+and+embryos+enables+a+precise+match+between+3D+virtual+histology,+classical+histology+and+immunochemistry.">&#956;CT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry.</a> PLoS One. 12 (2), e0170597 (2017).</li><li>Zikmund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-contrast+differentiation+resolution+3D+imaging+of+rodent+brain+by+X-ray+computed+microtomography.">High-contrast differentiation resolution 3D imaging of rodent brain by X-ray computed microtomography.</a> Journal of Instrumentation. 13 (2), 1-12 (2018).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+MicroCT+using+iodine-based+contrast.">A novel procedure for rapid imaging of adult mouse brains with MicroCT using iodine-based contrast.</a> PLoS One. 10 (11), e0142974 (2015).</li><li>Nieminen, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+histopathological+grading+of+osteochondral+tissue+using+contrast-enhanced+micro-computed+tomography.">3D histopathological grading of osteochondral tissue using contrast-enhanced micro-computed tomography.</a> Osteoarthritis Cartilage. 26 (8), 1118-1126 (2018).</li><li>Greef, D. D., Buytaert, J. A. N., Aerts, J. R. M., Hoorebeke, L. V., Dierick, M., Dirckx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+of+Human+Middle+Ear+Morphology+Based+on+Micro-CT+Imaging+of+Phosphotungstic+Acid+Stained+Samples.">Details of Human Middle Ear Morphology Based on Micro-CT Imaging of Phosphotungstic Acid Stained Samples.</a> Journal of Morphology. 276 (9), 1025-1046 (2015).</li><li>Sutter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast-Enhanced+Microtomographic+Characterisation+of+Vessels+in+Native+Bone+and+Engineered+Vascularised+Grafts+Using+Ink-Gelatin+Perfusion+and+Phosphotungstic+Acid.">Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid.</a> Contrast Media &amp; Molecular Imaging. 2017, (2017).</li></ol>

We implemented microCT with PTA preparation in the examination of human soft tissues. Briefly, specimens are harvested and fixed in formalin for a few days, followed by dehydration in serial ethanol solutions. Placing the sample into the PTA solution directly after formalin fixation can result in some tissue cracking due to rapid dehydration. Therefore, serial dehydration is needed before PTA staining. Next, the samples are stained using PTA solution for about a week. MicroCT scanning, 3D reconstruction, and analysis can...

Manual dissection and histological observation are typically used to examine human tissues, such as muscles and connective tissues. However, manual dissection can easily damage delicate structures, and histological observation provides limited information about flat cross-sectional surfaces1,2. Therefore, improved methods are needed to examine tissues more precisely and efficiently.
Conventional computed tomography (CT) is generally used in clinical practice, but it lacks the ability to distinguish small structures2,3. Micro X-ray CT (microCT) is an effective tool for obtaining three-dimensional (3D) information of small structures from specimens, without destroying them. However, microCT has limited applications because only dense tissues can be visualized clearly; it cannot be used to differentiate soft tissues. To overcome this limitation, staining agents can be used. Contrast-enhancing agents, like phosphotungstic acid (PTA), phosphomolybdic acid, and Lugol&#8217;s iodine, improve the soft tissue contrast rate during scanning4,5. Several studies comparing these agents suggest that PTA demonstrates good performance and is easy to handle6,7,8.
The orbicularis retaining ligament (ORL) is a delicate structure around the orbit, which can be easily damaged during conventional observation9. We examined and successfully retrieved 3D information on this structure using microCT with PTA as a contrast agent. This method can be applied to studies on other human tissues, such as the heart and liver, with appropriate modifications10,11,12.

<table>
	<tbody>
		<tr>
			<td>12 Tungsto(&#8549;)phosphoric acid n-hydrate 
			Phosphotungstic acid</td>
			<td>Junsei</td>
			<td>84220-0410</td>
			<td>PTA powder</td>
		</tr>
		<tr>
			<td>CTvox</td>
			<td>Bruker</td>
			<td>ver 2.7</td>
			<td>3D recon software</td>
		</tr>
		<tr>
			<td>Nrecon</td>
			<td>Bruker</td>
			<td>ver 1.7.0.4</td>
			<td>Reconstruction software</td>
		</tr>
		<tr>
			<td>Skyscan</td>
			<td>Bruker</td>
			<td>1173</td>
			<td>MicroCT scanner</td>
		</tr>
		<tr>
			<td>Tconv</td>
			<td>Bruker</td>
			<td>ver 2.0</td>
			<td>File resizing software</td>
		</tr>
	</tbody>
</table>

use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

The detailed reconstruction of the ORL was achieved by microCT with PTA preparation (Figure 4). The ligamentous fibromuscular structure extending obliquely between the dermis and periosteum was distinctly observed (Figure 4A). In the coronal view (Figure 4B), the amount and complexity of fibers increased laterally. In the horizontal view (Figure 4C), an elaborate meshwork with an arborized formation was...

Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

We implement microCT with phosphotungstic acid to enhance the soft tissue contrast in the non-invasive investigation of the human orbicularis retaining ligament, a delicate structure within the orbit area. This protocol has been optimized to aid researchers in the non-invasive investigation of human soft tissues and is simple to perform with a reasonable amount of expense. To obtain the samples, first use a colored pencil to indicate the cutting area on the skin for the sample harvesting.Confirm that the marked line extends medially to a medial campus, laterally to a lateral campus, superiorly to a superior border of the lower eyelid, and inferiorly to one centimeter below the line from the orbital rim. Next, use a blade to cut the facial tissues along the marked line, making sure that the cut is deep enough for the blade tip to touch the bone. The harvested sample must include the skin, subcutaneous tissue, muscle, fat, and periosteum.Upon collection, immediately fix the sample in 10%formalin for five to seven days at room temperature. At the end of the fixation period, slice the sample in three five to seven-millimeter thick pieces. Use a needle and black thread to sew the superolateral side of each sample such that the direction of the sample can be determined at later time points.Dehydrate the sample in an ascending ethanol concentration series for one day per concentration, leaving the samples in the 70%ethanol solution until they're staining. One week before the microcomputer tomography scanning is scheduled, add 2.1 grams of PTA to 210 milliliters of 70%ethanol and place the solution on a shaker at 55 to 60 rotations per minute. Prepare three 70-milliliter plastic containers for each sliced piece.When the dye has dissolved into the alcohol solution, fill the containers with the solution. Place one specimen into each container of 1%PTA, and shake the samples at 55 to 60 rotations per minute for five to seven days. When the staining is complete, store the samples in fresh 70%ethanol for one week to prepare for the scanning.For microCT scanning, wrap the samples in Parafilm to prevent drying and place a sample onto the scanner tray. Set the scanner's source voltage to 70 kilovolts, the source current to 114 microamps, the aluminum filter to 0.5 millimeters, the image pixel size to 20 squared micrometers, the pixels to 2, 240 by 2, 240, the exposure to 500 milliseconds, and the rotation step to 0.3 degrees. Then, initiate the scanning.When the scanning is complete, open the reconstruction software and select Actions and Open Dataset to launch the scanned files. Select the Settings tab and set the ring artifacts reduction to seven and the beam-hardening correction. To begin the reconstruction, select Start.The final data will be stored in the designated folder. At the end of the reconstruction, open the file resizing software, and select Source data set to launch the reconstructed files. Select jpg on the Destination data set tab and select the 1/2 Resizing option with the Quality option of No interpolation Then adjust the slide bar to 100 in the Image compression tab and start the conversion.For 3D reconstruction, open the 3D volume rendering software, and select Actions and Load Volume Data. To adjust the brightness and contrast level, modify the shape transfer function in the histogram in the Transfer Function Editor tab. Next select Options and Lighting, and select the Shadows and Surface Lighting icons to achieve a realistic modeling tone.Click and drag, and right-click and drag to move and rotate the 3D image to find the best view, scrolling to zoom in or out as desired. Click Shift and drag to slide the place to view the sectional images. Turn on the Light icon and adjust the lighting indication bar to find the best lighting for viewing, then turn off the Light icon and close the Lighting tab.In the Options tab, select Show and Clipping Box to hide the box for the final image. Then, select Actions and Save Image to store the image. This detailed reconstruction of the ORL was achieved by microCT with PTA preparation as demonstrated, allowing visualization of a ligamentous fibromuscular structure extending obliquely between the dermis and the periosteum.In the coronal view, the amount and complexity of the fibers increases laterally. In horizontal view, an elaborate meshwork with an arborized formation is observed. In the sagittal view, the thicknesses of the ORL fibers decreases inferiorly.Overall, this multi-directional observation proves that the ORL is made up of a multilayered meshwork of continuous plates with variations in the number and thickness of the fibers depending on their location. When the duration of the staining is insufficient compared to the volume of a specimen, the final image may include an empty hole in the central area of the specimen. Other agents beside PTA can be used and different agents have different merits or staining features interests.Therefore, it is useful to optimize the staining region according to your experiment.

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Research

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Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...