This work was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health through Award No. UL1 TR000042 and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health through Award No. R21 EB018432.

1. Download STL Models from the NIH 3D Print Exchange
<ol>
	<li>Navigate to the NIH 3D Print Exchange (3dprint.nih.gov) and in the Search entry type Simulated Aneurysmal Aortic Phantom Mold and hit the enter key.</li>
	<li>In the subsequent list returned from the search, find the model &#34; 3DPX-009210&#34; and click that entry.</li>
	<li>Click the download button and subsequently click the Simulated Aneurysmal Aortic Phantom Mold.zip file from the drop-down list to download this file.</li>
	<li>Double click the downloaded file to unzip it and store the resulting files (InnerDistSTL.stl, InnerProxSTL.stl, OuterAntSTL.stl, OuterPostSTL.stl, BackgroundMoldSTL.stl and SampleMoldSTL.stl) to the computer used for 3D printing in Steps 2.1-2.7. 
	NOTE: One may alternatively download each of the files listed in Step 1.4 separately.</li>
</ol>
2. 3D Printing of Molds
<ol>
	<li>Open the 3D printer interface software and use the Connect button to connect to the printer.</li>
	<li>Import the downloaded STL file OuterAntSTL.stl (Figure 1a, blue) into the 3D printing software. In the 3D printing software, select the Edit button and orient the mold part by clicking the Rotate menu and then clicking the X, Y, or Z buttons to align the long axis parallel to the print bed with the outside of the mold facing the print bed. Click the Save button and then click the Print button and print the mold part using polylactic acid (PLA) plastic filament on a single extruder.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57984/57984fig1.jpg" /> 
Figure 1: CAD Representation of Phantom, Background and Sample Molds. (a) - (b) 3D CAD images of vessel mold and orientation of parts for assembly. Registration spacers (i), pins (ii), holes (iii) and the fill hole are shown. (c) Drawing of inner lumen highlighting the inner vessel dimensions. (d) CAD rendering of the sample molds. (e) CAD rendering of the background phantom mold. <a href="https://www.jove.com/files/ftp_upload/57984/57984fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Repeat Step 2.2 for the OuterPostSTL.stl file (Figure 1a, red).</li>
	<li>Following the same process from Step 2.2, import the STL file InnerDistSTL.stl (Figure 1a, white) into the 3D printing software and select the &#34;Edit&#34; button and in the Rotate menu click the X, Y, or Z buttons to align the long axis perpendicular to the print bed and such that the registration pin (i) is in contact with the print bed. Click the Save button and then click the Print button and print the mold part using PLA plastic filament on a single extruder. 
	NOTE: Do not print this part with the support structure. Do not use more than 30% infill for this printed part.</li>
	<li>Import the STL file SampleMoldSTL.stl (Figure 1d) into the 3D printing software. Select the Edit button and in the Rotate menu click the X, Y, or Z buttons to align the part such that the inside of the mold is facing up from the print bed. Click the Save button and then click the Print button and print the mold part using PLA plastic filament on a single extruder. 
	NOTE: Do not print this part with the support structure. Print 3 or more sample molds.</li>
	<li>Import the STL file BackgroundMoldSTL.stl (Figure 1e) into the 3D printing software. Select the &#34;Edit&#34; button and in the Rotate menu click the X, Y, or Z buttons to align the part such that the bottom of the mold (i.e., the closed end of the cylinder) is facing the print bed. Click the Save button and then click the Print button and print the mold part using PLA plastic filament on a single extruder. 
	NOTE: Do not print this part with the support structure.</li>
	<li>Import the STL file InnerDistSTL.stl (Figure 1a, yellow) into the 3D printing software. Select the &#34;Edit&#34; button and in the Rotate menu click the X, Y, or Z buttons to align the part such that the long axis is perpendicular to the print bed and bifurcation registration pins (i) are facing the print bed. Click the Save button and then click the Print button and print the mold part using polyvinyl acid (PVA) plastic filament on a single extruder.</li>
	<li>Remove any support material from the 3D printed parts of Steps 2.1-2.7 (Figure 2a). 
	NOTE: It is not necessary to remove the support structure from the outer mold parts if they do not interfere with the mold assembly.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57984/57984fig2.jpg" /> 
Figure 2: Vessel Phantom Mold Assembly and Final Vessel Phantom. (a) The final printed mold of inner and outer lumen molds. The distal end of the inner lumen printed in a dissolvable PVA plastic and is attached to the proximal end of the inner lumen mold using deformable wax. (b) Tubing attached to the injection port of the outer lumen mold and the syringe stopper. (c) The inner lumen mold after spray coating of flexible sealant. (d) Assembly of bulge side of the outer lumen mold and inner lumen mold with PVA-c (dyed red) added for stiff aneurysm phantoms. (e) Full vessel mold assembled and clamped. (f) Deformable wax applied to the seams of the outer lumen mold to prevent PVA-c from leaking from the mold. (g) Final PVA-c phantom after 5 freeze/thaw cycles and removal from the mold. <a href="https://www.jove.com/files/ftp_upload/57984/57984fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Hydrogel Preparation
<ol>
	<li>Mix 22.2 g of PVA-c powder in 200 mL of tap water (10% by mass) in a glass beaker. Microwave the solution to a boil and stir. Repeat this step until all of the PVA powder is dissolved and the solution appears translucent.</li>
	<li>Suspend 0.4 g of calcium carbonate powder (0.2% by mass) in 10 mL of water and add to the solution from Step 2.1 to act as ultrasound scatterers. Mix Thoroughly. Cover the solution and allow it to cool to the room temperature (RT). 
	NOTE: For homogeneous phantoms skip to Step 3.5</li>
	<li>Mix 17.6 g of PVA-c powder in 100 mL of tap water (15% by mass or as desired) in a separate glass beaker. Microwave the solution to a boil and stir. Repeat this step until all the PVA powder is dissolved and the solution appears translucent.</li>
	<li>Suspend 0.4 g of calcium carbonate powder (0.2% by mass) in 5 mL of water and add to the solution from Step 2.3. Mix Thoroughly. Cover the solution and allow it to cool to the RT.</li>
	<li>Mix 183.7 g of PVA-c powder in 3.5 L of tap water (5% by mass) in a separate large pot. Bring the solution to a boil and stir. Remove the pot from the heat once the PVA powder is dissolved and the solution appears translucent.</li>
	<li>Suspend 7.4 g of calcium carbonate powder (0.2% by mass) in 10 mL of water and add to the solution from Step 2.5. Mix Thoroughly. Cover the solution and allow it to cool to the RT.</li>
</ol>
4. Assembly of Molds
<ol>
	<li>Attach approximately 100 mm of the flexible tubing to the injection port of the outer lumen mold. To the opposite end of the tubing, attach a stopcock with syringe connections (Figure 2b).</li>
	<li>Align the registration pins of the inner lumen mold and, using deformable wax, adhere the bulging vessel part of the inner lumen mold to the straight vessel part of the inner lumen mold.</li>
	<li>In a well-ventilated area, apply a spray-on flexible rubber coating to the aneurysmal end of the inner lumen mold to prevent the hydrogel from dissolving the PVA mold part during the molding process (Figure 2c). 
	NOTE: For homogeneous phantoms skip to Step 4.6.</li>
	<li>With the larger side of the aneurysmal part of the outer mold facing down, fill the bulge with 15 mL of the solution created in Steps 3.3-3.4 (Figure 2b,). Place the assembled inner mold parts in the front outer mold part (Figure 2d). Use rubber bands to hold the inner lumen part in place. 
	NOTE: In Figure 2, PVA-c is dyed red for visibility.</li>
	<li>Freeze the mold assembly in a -20 &#176;C freezer for 12 h and remove from the freezer. Move on to Step 4.6 without letting the solution in the mold assembly thaw.</li>
	<li>While waiting for the mold to freeze (Step 4.4), apply a generous amount of deformable wax to the Back surface of a printed sample mold and clamp it to a flat plastic sheet cut to the minimum size of approximately 100 mm by 60 mm by 10 mm (Figure 3a). Fill the space between the mold and the plastic sheet with the same PVA solution used in Step 4.3. Freeze the sample mold in the same freezer (-20 &#176;C) as the vessel mold in Step 4.4.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57984/57984fig3.jpg" /> 
Figure 3: Sample mold and Final Sample and Background Phantoms. (a) Clamped sample mold and clear plastic sheet. PVA-c is poured into the sample mold and air bubbles are allowed to surface. (b) PVA-c sample after final freeze/thaw cycle. (c) Experimental US imaging setup of phantom attached to simulator pump and placed in the background PVA-c phantom. <a href="https://www.jove.com/files/ftp_upload/57984/57984fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Assemble and clamp together the entire vessel mold in the orientation shown in Figure 1a and 1b (Figure 2e). Line the seams of the outer lumen molds using a deformable wax to ensure that the hydrogel does not leak during injection (Figure 2f).</li>
	<li>Fill a 60 mL syringe with the PVA-c solution made in Steps 3.1 and 3.2. With the bifurcation end of the mold up inject the PVA-c solution into the mold assembly avoiding air bubbles in the injected solution. 
	NOTE: If any leaks occur during the injection, pause injection and patch leaky areas with deformable wax. Repeat syringe injections until the PVA-c solution fills the mold.</li>
	<li>Allow the mold to sit for 30 min, tapping the mold gently every 10 min to allow any air bubbles to rise to the top of the mold. Repeat the syringe injection if needed to top off the mold. Freeze the entire mold assembly for 12 h and remove from the freezer. Allow the mold assembly to thaw at RT for 12 h.</li>
	<li>While waiting for the mold to freeze (Step 4.8), assemble and clamp another sample mold and flat plastic sheet cut as described in Step 4.5 (Figure 3a). Fill the space between the mold and the plastic sheet with the same PVA solution used in Step 4.7. Freeze and thaw the sample mold in the same freezer (-20 &#176;C) and at the same time as the vessel mold in Step 4.8 and the sample mold of Step 4.5.</li>
	<li>Freeze and thaw the vessel mold and both the sample molds from Steps 4.5, 4.8 and 4.9 four more times, for a total five 24 -h freeze/thaw cycles. After the 5th freeze/thaw cycle, remove the PVA-c testing samples from their molds (Figure 3b). Trim any excess cryogel from the samples and store them in a sealed container of a 5% by volume bleach/water solution at RT.</li>
	<li>Remove the PVA-c vessel from the outer lumen mold. Carefully separate the straight vessel part of the inner lumen mold from the aneurysmal part and remove from the PVA-c vessel. Cut the registration spacers from the bifurcated end of the aneurysmal part of the inner lumen mold to expose the printed PVA filament. Place in a water bath at RT to dissolve the PVA aneurysmal part. 
	NOTE: This may take 24 h or more, however, adding warm water to the bath may speed the dissolving process.</li>
	<li>After dissolving and removing the PVA printed part from inside of the vessel phantom, store the phantom in a sealed container of a 5% by volume bleach/water solution at RT.</li>
	<li>Fill the background mold with approximately 3.3 L of the PVA-c solution made in Steps 3.5 and 3.6. Freeze (-20 &#176;C) the background mold for 12 h and remove from the freezer. Allow the mold to thaw at RT for 12 h and repeat for a total of 2 freeze/thaw cycles.</li>
	<li>At the same time as Step 4.13, fill a sample mold assembly with the same PVA-c solution used in Step 4.13 and put it through the same freeze/thaw samples as the background mold.</li>
	<li>After the 2nd thaw, remove the background sample and background phantom from their molds and store them in a sealed container of a 5% by volume bleach/water solution at RT.</li>
</ol>
5. Phantom and Sample Testing
<ol>
	<li>Place the vessel phantom and background phantom into a large water bath. Attach the larger vessel end to the output of the hemodynamic water pump42,43 using tubing clamps (Figure 3c). Place the vessel phantom in the background phantom and then attach the bifurcated ends of the phantom to the inlet to the hemodynamic pump using tubing clamps.</li>
	<li>Place a solid-state pressure sensor catheter in the system of the vessel and pump near the inlet of the hemodynamic pump. Run the hemodynamic pump such that the pressures of the wall deformations are between a minimum of 0 kPa and a maximum 7.5 kPa (Figure 4a).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57984/57984fig4.jpg" /> 
Figure 4: Imaging Protocol. (a) Pressure profile measured during the phantom imaging setup. (b) A representative B-mode image of the phantom at the minimum pressure. (c) B-mode at maximum pressure. <a href="https://www.jove.com/files/ftp_upload/57984/57984fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Use an ultrasound (US) system and a convex transducer with a center frequency of approximately 5 MHz to collect US Images of the background and vessel phantoms in cross-section at the location of the maximum vessel diameter (Figure 4b and 4c). Record the pressure data using a digital acquisition system (Figure 4a). 
	NOTE: Details for performing the image acquisition in this step can be found in Mix et al44.</li>
	<li>Obtain the displacement estimations by using a non-rigid image registration-based technique as described in Mix et al.44. From the measurements of the two-dimensional (2D) displacement field (ui(x)), calculate the 2D strain tensor field (&#949;ij(x)) by evaluating the symmetric part of the gradient of the displacement field: 
	<img alt="Equation 1" src="/files/ftp_upload/57984/57984eq1.jpg" /></li>
	<li>Then, calculate the maximum principal strain (&#949;p) as the maximum principal component of the strain tensor field using the following equation: 
	<img alt="Equation 2" src="/files/ftp_upload/57984/57984eq2.jpg" /></li>
	<li>Lastly, determine the frame of the principal strain at the peak pressure and divide this strain tensor field by difference in the maximum and minimum catheter measured pressures (Figure 4a), or the pulse pressure (PP), to spatially resolved pressure normalized principle strain (&#949;p/PP).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57984/57984fig5.jpg" /> 
Figure 5: Pressure Normalized Strain Images. Representative images of the pressure normalized strain (&#949;p/PP) in%/kPa measured within the vessel for the homogeneous 10% by mass vessel phantom (a) and the heterogeneous phantoms with a 15% by mass (b), 20% by mass and 25% by mass anterior aneurysmal section (top of vessel). This figure has been modified from Mix et al.44. <a href="https://www.jove.com/files/ftp_upload/57984/57984fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors have nothing to disclose.

This paper presents a technique to manufacture tissue-mimicking phantoms for the use in testing elastography or elasticity imaging algorithms. The combined use of CAD and 3D printing allows for efficient design of aortic mimicking phantoms with complex geometries, beyond tubular phantoms, including aneurysmal bulges. The creation of the phantom is done in 4 steps; 1) design of the phantom geometry, 2) printing of the phantom mold parts, 3) mixing of the cryogel solutions which will ultimately mimic the ultrasonic characteristics and mechanical properties of the phantom vessels and 4) pouring/injecting of the cryogel solution into the mold, setting the PVA-c with freeze-thaw cycles and removal of the phantom from the mold. The use of CAD in the in the design of the molds obtained in Step 1 allows for a simple means to precisely modify the geometry of the phantoms. Printing of the mold parts currently takes approximately 5-8 h depending on the size of the print and thus can easily be made for repeated modifications to the molds.
In Step 3, the cryogel solutions are created to mimic the vessel, aneurysm and background tissue with the calcium carbonate particles mimicking the US scatter of the tissue. The cryogel solutions should be stirred prior to use if the calcium particles have settled out of the mixture. The exact concentration of the mixture of the cryogel will determine the final mechanical properties of the phantoms. Thus, it is important to create the independent samples of each of the solutions used in the phantom vessel and background. Although not part of the protocol here, independent measurements of the sample&#39;s elastic modulus should be obtained using uniaxial tension testing. Independent mechanical testing of the PVA-c samples for the 10%, 15%, 20% and 25% phantoms created in the representative results had measured shear modulus of 17.4 &#177; 1.0 kPa, 48.3 &#177; 5.7 kPa, 95.1 &#177; 0.4 kPa and 170.0 &#177; 4.1 kPa, respectively.
Step 4 is the most critical step in creating these phantoms. Although the registrations pins are in place to keep the mold parts in their proper positions relative to the others, it is important to ensure that the mold parts do not separate during the molding process. Thus, the use of the clamps to hold the mold. The most important consideration of Step 4 is to minimize air bubbles trapped in the mold prior to the first freeze-thaw cycle. It is often useful to disassemble one side of the outer mold and inspect the phantom after the first freeze-thaw cycle to ensure it formed properly. This can save time wasted putting a &#34;bad&#34; phantom through additional cycles. Once the phantom has been removed from the mold entirely, it can be stored in water for several weeks with continued use.
The PVA-c phantoms developed in this work were created to specifically mimic the ultrasonic and material stiffness of aortic tissue. The use of polyvinyl alcohol cryogel allows for a wider range of possible mechanical stiffness, to better mimic the changing material properties of aortic tissue compared to more rubber like materials33,34. In addition, the use of hydrogel and investment casting better captures the acoustic properties of casted rubbers or directly 3D printed materials33,45. Some air bubbles can get trapped in our molds prior to the first freeze-thaw cycle. This can cause gaps in the phantom and lead to material weakness or acoustic artifacts. Thus, it is recommended to inspect phantoms out of the mold after the first freeze-thaw to determine if the process should be restarted. In addition, the authors have found that the inner mold can sometimes shift during the freezing of the aneurysmal portion of the phantoms. If this occurs, one modification of the above protocol would be to create a 3D printed, or otherwise designed, part to firmly hold the inner lumen mold to the anterior outer mold during the freezing of this section. The authors have found that using the posterior side of the outer mold and a 5 mm spacer between the posterior outer mold and the inner mold works well for this purpose.
The phantom developed here is ideal for studying the influence of changes in aneurysmal diameter, and luminal thickness or potentially the presence of thrombus in the tissue by editing the original CAD files. However, previous work has also shown that this manufacturing technique can be modified to produce patient-specific phantom geometries using computed tomography images and segmentation software, rather than CAD design, to create the 3D printed phantom molds44. The results shown here demonstrate that the algorithm was able to visualize the manufactured variations in the mechanical properties of the phantom sections. It should be noted that although these phantoms were used to test US-based imaging techniques, they are also compatible with magnetic resonance and computed tomography imaging systems and that they may also be used beyond the purpose of elasticity imaging, for a wide range of novel imaging techniques and modalities.

Abdominal aortic aneurysms (AAA) are focal enlargements of the aorta that occur preferentially near the aortic bifurcation1. The exact cause of AAA formation is unknown, although many theories suggest that the pathogenesis is multifactorial, with genetic, behavioral, hemodynamic, and environmental factors contributing2,3. While the diagnosis of an abdominal aortic aneurysm can be obtained using non-invasive imaging techniques, the prediction of patient-specific rupture risk is not as precise4,5,6. Surgical repair can reduce the risk of aortic rupture, but operative repair of the aorta carries a high rate of associated morbidity and mortality7. Current surgical practices use the &#34;maximum size criteria&#34;, or maximum absolute diameter of the aneurysms, to predict a patient&#39;s risk of rupture. Unfortunately, it has been well established that an aneurysm still ruptures below sizes clinically acceptable for surgical repair, meaning that patients with any sized aneurysm carry some risk of rupture8,9,10,11,12,13. Additionally, it is known that historical reports of rupture risk are likely over-estimations of the true rupture risk, meaning many patients are exposed to surgical risk without benefit13. A more accurate assessment of the patient-specific rupture risk is needed to help stratify a patient&#39;s risk-benefit ratio of undergoing surgical aneurysm repair.
It has been shown that the spatial stress distribution within an AAA is of critical importance in determining rupture potential and may be a better indicator than maximum diameter14,15,16,17,18. Most of the recent studies that investigate the mechanics of AAA rupture use segmented geometries from X-ray computed tomography (CT) images, and population averaged mechanical properties of aortic tissue measured ex vivo. Finite element (FE) models are then used to predict the vessel wall stresses14,15,16,17,18. However, because the mechanical properties are determined following the tissue excision, it is unclear whether the resulting models accurately depict the resulting in vivo patient-specific stresses. These studies typically assume homogeneous vessel wall material properties and don&#39;t account for the highly heterogeneous structure of the aortic wall and thrombus19,20,21,22,23,24,25.
Ultrasound-based elasticity imaging is used clinically to diagnose and monitor a variety of disease pathologies26. This technology provides a non-invasive means to interrogate the physical interactions of soft tissues. Vascular US elasticity imaging has been used as an adjunct imaging modality to clinical US evaluation in the screening and monitoring of AAAs. The combination of these techniques provides both geometric information, such as diameter and length, as well as mechanical data, such as relative stiffness and stiffness variation. While many elasticity imaging techniques require an external load to induce a measurable tissue deformation, the tissue motion to be measured here is induced by changes in the aortic pressure caused by the beating heart. Numerous methods have been published to spatially resolve strain fields in deforming vessels, however, validation studies of these methods have been limited to human patients, animal models, or ex vivo tissue samples27,28,29,30,31,32. To date, few methods allow for creations of custom geometries with spatially varied material properties27,29.
Here we present a method of manufacturing US compatible, tissue-mimicking phantoms that can be tailored to a variety of relevant aortic geometries and material properties for validation of US elastography techniques. Although previous groups have been able to design complex geometry phantoms to mimic AAA geometries using 3D printing technology33,34, printable rubbers are known to have a high attenuation to US and do not have a means to later their material properties. Phantoms are made from polyvinyl alcohol cryogel (PVA-c), which has been previously shown to be ideal for mimicking vascular tissue properties35. These phantoms can be used in US, magnetic resonance, and elastographic imaging36,37,38. The aortic aneurysm geometry was designed similarly to that of the simulation model created by Vorp et al.14. The vessel has a nominal diameter of 22.5 mm and has an aneurysmal bulge that is 64 mm bulge long, 47 mm in diameter and eccentric (&#946; = 0.6)14 to the anterior side of the phantom. The last section mimics the iliac bifurcation with a distal diameter of 15 mm. The phantom was chosen to have a constant thickness of approximately 5 mm. Raghavan et al. reported in a small study that the vessel thickness of AAA ranges from 0.23-4.26 mm, with a median value of 1.48 mm39. A nominal vessel thickness on the larger end of that spectrum was chosen here for manufacturing concerns with the expectation that improved 3D printing techniques will improve the minimum phantom thickness that is able to be molded. Phantom molds were designed in CAD and are 3D printed using commercially available printers and filament.
The molds are injection filled with the PVA-c solution and subjected to a series of freeze/thaw cycles (-20 &#176;C and +20&#176;C) to cross-link the PVA-c polymer and polymerize the gel. The elastic modulus of the PVA-c is controlled by altering the concentration of the PVA-c gel or the number of freeze-thaw cycles. The aneurysmal section of the phantom required loss mold to remove from the inner lumen of the vessel. This was accomplished by the use of a polyvinyl alcohol, 3D printer filament (PVA). Although chemically similar to the PVA-c powder, the PVA filament does not polymerize when frozen and, as such, can be dissolved in water after the PVA-c has been set. Additional sample molds are printed to create tensile testing specimens, in a &#34;dog bone&#34; configuration, with the same PVA-c concentration. These molds undergo the same freeze/thaw cycles and are used for tensile testing to independently measure the elastic modulus of the phantom sections. A background material was manufactured with softer PVA-c, made to simulate tissues of the retroperitoneum40,41. This background phantom was manufactured as a homogeneous axisymmetric cylindrical tube with a 4 cm inner diameter, a 16.5 cm outer diameter, and a length of 16.5 cm. It was made from a 5% PVA solution and subjected to a total of two freeze-thaw cycles.
The final AAA phantoms were placed in the background phantom and connected, via tube fittings and clamps, to a hemodynamic water pump designed to deform the phantoms with physiologic cyclic flows and pressures. The pump speed was set to deliver approximately a 6-7 kPa pressure pulse at a rate of approximately 1 Hz. Ultra sound image sequences of the deforming phantoms were collected, and the pressure normalized strain was calculated to identify differences in the spatially varied mechanical properties. Representative results of the pressure normalized strain images within the vessel region are presented. The increasing regional differences in the normalized strain of the stiffer heterogenous phantoms, relative to the homogeneous phantom, demonstrate the differences in the vessel stiffness and our ability to measure it.

<table><tbody><tr><td>PLA filament</td><td>MatterHackers, MatterHackers.com</td><td>MEEDKTKU</td><td></td></tr><tr><td>PVA filament</td><td>MatterHackers, MatterHackers.com</td><td>M4MJTECR</td><td></td></tr><tr><td>LeakSeal</td><td>RPM International Inc., Rustoleum.com</td><td>265495</td><td></td></tr><tr><td>Polyvinyl alcohol powder (Elvanol 71-30)</td><td>DowDuPont Inc., ChemistryStore.com</td><td>SKU: 81015</td><td></td></tr><tr><td>Calcium Carbonate Powder</td><td>greenwaybiotech.com via amazon.com</td><td>Amazon: B00HFFCBYQ</td><td></td></tr><tr><td>Tacky Wax</td><td>bards.com via amazon.com</td><td>Bards: BB759&lt;br/&gt; Amazon: B016KBDYRS</td><td></td></tr><tr><td>Rostock max 3D Printer</td><td>SeeMeCNC, seemecnc.com</td><td>SKU: 84459</td><td></td></tr><tr><td>Onshape CAD software</td><td>OnShape, onshape.com</td><td></td><td></td></tr><tr><td>Mattercontrol printer software</td><td>MatterHackers, MatterHackers.com</td><td></td><td></td></tr><tr><td>Mikro-Cath pressure catheter and device</td><td>Millar, Inc., millar.com</td><td>4501016/B</td><td></td></tr><tr><td>BNC digital acquisition</td><td>National Instruments Corporation, ni.com</td><td>NI USB-6251 BNC</td><td></td></tr><tr><td>clear cast acrylic sheet</td><td>mcmaster-carr Supply Company, mcmaster.com</td><td>8560K274</td><td></td></tr><tr><td>Cole-Parmer Stopcocks with Luer Connections; 3-way; male lock, Non-sterile</td><td>Cole-Parmer, coleparmer.com</td><td>EW-30600-02</td><td></td></tr><tr><td>BD Disposable Syringes (60 mL, Luer lock)</td><td>Cole-Parmer, coleparmer.com</td><td>EW-07945-28</td><td></td></tr><tr><td>6 Inch Ratchet Bar Clamp / 12 Inch Spreader</td><td>Tekton, Inc., www.tekton.com</td><td>39181</td><td></td></tr><tr><td>Tygon PVC Clear Tubing</td><td>mcmaster-carr Supply Company, mcmaster.com</td><td>6516T53</td><td></td></tr><tr><td>MTS Qtest Q/5</td><td>MTS Systems Corperation, www.mts.com</td><td>4501016</td><td></td></tr><tr><td>MTS 5N Load Cell</td><td>MTS Systems Corperation, www.mts.com</td><td>4501016/B</td><td></td></tr><tr><td>Abaqus FEA</td><td>Dassault Syst&amp;egrave;mes, 3ds.com</td><td></td><td></td></tr></tbody></table>

manufacturing abdominal aorta hydrogel tissue-mimicking phantoms for ultrasound elastography validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Representative B-mode images of the vessel mimicking phantoms are shown for the minimum and maximum pressures measured by the catheter (Figure 4b and 4c, respectively). The pressure-normalized strain (&#949;p/PP) in%/kPa is shown for four different manufactured phantoms (Figure 5). Figure 5a shows the measured pressure-normalized strain within a homogeneous phantom manufactured with a 10% by mass PVA-c solution. The ratio of the average strain measured within the posterior quarter (image bottom) of the phantom to the average strain in the anterior quarter (image top) was 0.92. Figure 5b shows &#949;p/PP for a phantom in which the aneurysmal section of the phantom was manufactured with a 15% by mass PVA-c solution and the remainder of the phantom was made using the 10% by mass PVA-c. The ratio of posterior to anterior strain for this phantom was found to be 1.87. Figure 5c shows &#949;p/PP for the heterogeneous phantom with 20% by mass PVA-c, with a posterior to anterior strain ratio of 4.23. Figure 5d shows &#949;p/PP for the heterogeneous phantom with 25% by mass PVA-c, with a posterior to anterior strain ratio of 7.37.
The results presented here show that abdominal aortic phantoms were created with complex geometries and spatially varying material properties. Design of phantom geometries were, or more specifically, phantom molds were done using computer software which facilitates phantom geometry alterations (Figure 1a and 1b). Molds can be readily 3D printed and assembled and complex mold geometries can be printed using PVA filament and removed, similar lost-wax casting techniques. The final vessel phantoms can be dynamically pressurized and are stable under large loads (Figure 4a). Phantoms are compatible with ultrasound imaging (Figure 4b and 4c) and have material properties mimicking abdominal aortic stiffnesses. Variations in the strain ratios in the anterior to posterior regions of the strain images demonstrate that the regions have varying material properties (Figure 5) and the independent mechanical testing on the samples quantify the exact values of their respective shear moduli.

Watch this Scientific Journal Video about Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation at JoVE.com

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...

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Research

JoVE Journal

Bioengineering

15.3K Views.  University of Rochester Medical Center. Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Video: Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of an optical imaging device are greatly affected by the performance characteristics of its components, the test environment, and the operations. Therefore, it is necessary to calibrate these devices by traceable phantom standards. However, most of the currently available phantoms are homogeneous phantoms that cannot simulate multimodal and dynamic characteristics of biological tissue. Here, we show the fabrication of heterogeneous tissue-simulating phantoms using a production line integrating a spin coating module, a polyjet module, a fused deposition modeling (FDM) module, and an automatic control framework. The structural information and the optical parameters of a &#34;digital optical phantom&#34; are defined in a prototype file, imported to the production line, and fabricated layer-by-layer with sequential switch between different printing modalities. Technical capability of such a production line is exemplified by the automatic printing of skin-simulating phantoms that comprise the epidermis, dermis, subcutaneous tissue, and an embedded tumor.

Spin coating, polyjet printing, and fused deposition modeling are integrated to produce multilayered heterogeneous phantoms that simulate structural and functional properties of biological tissue.

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of ...

Engineering

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Immunology and Infection

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

Medicine

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training ...

Fabrication of a Stable, Biologically Relevant Phantom Material

A Stable Phantom Material for Optical and Acoustic Imaging

Establishing tissue-mimicking biophotonic phantom materials that provide long-term stability are imperative to enable the comparison of biomedical imaging devices across vendors and institutions, support the development of internationally recognized standards, and assist the clinical translation of novel technologies. Here, a manufacturing process is presented that results in a stable, low-cost, tissue-mimicking copolymer-in-oil material for use in photoacoustic, optical, and ultrasound standardization efforts.
The base material consists of mineral oil and a copolymer with defined Chemical Abstract Service (CAS) numbers. The protocol presented here yields a representative material with a speed of sound c(f) = 1,481 &#177; 0.4 m&#183;s-1 at 5 MHz (corresponds to the speed of sound of water at 20 &#176;C), acoustic attenuation &#945;(f) = 6.1 &#177; 0.06 dB&#183;cm-1 at 5 MHz, optical absorption &#181;a(&#955;) = 0.05 &#177; 0.005 mm-1 at 800 nm, and optical scattering &#181;s&#39;(&#955;) = 1 &#177; 0.1 mm-1 at 800 nm. The material allows independent tuning of the acoustic and optical properties by respectively varying the polymer concentration or light scattering (titanium dioxide) and absorbing agents (oil-soluble dye). The fabrication of different phantom designs is displayed and the homogeneity of the resulting test objects is confirmed using photoacoustic imaging.
Due to its facile, repeatable fabrication process and durability, as well as its biologically relevant properties, the material recipe has high promise in multimodal acoustic-optical standardization initiatives.

Author Spotlight: A Stable Phantom Material for Optical and Acoustic Imaging  

This protocol describes the fabrication of a stable, biologically relevant phantom material for optical and acoustic biomedical imaging applications, featuring independently tunable acoustic and optical properties.

Establishing tissue-mimicking biophotonic phantom materials that provide long-term stability are imperative to enable the comparison of biomedical imaging ...

Building Custom Ballistic Gel Ultrasound Phantoms for Nerve Block Simulations

Building Affordable, Durable, Medium-Fidelity Ballistic Gel Phantoms for Ultrasound-Guided Nerve Block Training

Ultrasound phantoms - alternatives to live human tissue - give learners the opportunity to practice ultrasound-guided regional anesthesia without introducing undue risk to patients. Gelatin-based phantoms provide educators with durable and reusable task trainers; however, commercially available gel-based phantoms are expensive. Here, we investigate the production of durable, low-cost, ballistic gel-based ultrasound phantoms for median, femoral, suprainguinal fascia iliaca plane, and serratus anterior plane nerve blocks, as well as a methodology for producing a phantom for any ultrasound-guided nerve block procedure.
Computer-aided design (CAD) software was utilized to design four phantoms replicating the anatomy of median, femoral, suprainguinal fascia iliaca plane, and serratus anterior plane nerve blocks, including relevant landmarks and tissue planes. Plastic models of the desired tissue planes were 3D printed and used to create silicone molds. Ballistic gel was melted and mixed with flour and dye to create a liquid, echogenic ballistic gel, which was poured into the silicone molds. Vessels were simulated by creating negative space in the ballistic gel using metal rods. Nerves were simulated using yarn submerged in ultrasound gel. Simulated bones were designed using CAD and 3D printed.
Ballistic gel is a versatile, durable medium that can be used to simulate a variety of tissues and can be melted and molded into any shape. Under ultrasound, these phantoms provide realistic tissue planes that represent the borders between different layers of skin, muscle, and fascia. The echogenicity of the muscle tissue layers, nerves, vessels, and bones is realistic, and bones have significant posterior shadowing as would be observed in a human subject. These phantoms cost $200 each for the first phantom and $60 for each subsequent phantom. These phantoms require some technical skill to design, but they can be built for just 4% of the cost of their commercial counterparts.

Author Spotlight: Developing Cost-Effective and Durable Ultrasound-Guided 3D-Printed Nerve Block Trainers

Here, we demonstrate the design and creation of four custom ballistic gelatin ultrasound phantoms for ultrasound-guided regional anesthesia training. We designed the phantoms using computer-aided design software, used 3D-printed models to create silicone molds, and then poured melted ballistic gel into the molds to create custom tissue layers.