The authors thank Daniil Nikitichev and Steffi Mendes for their advice on using Meshmixer and Fernando Perez-Garcia for his advice on using 3D Slicer and for providing us code to automate some of the processing steps.
This work was supported by Wellcome Trust [203145Z/16/Z; 203148/Z/16/Z; WT106882], EPSRC [NS/A000050/1; NS/A000049/1], MRC [MC_PC_17180] and National Brain Appeal [NBA/NSG/SBS] funding. TV is supported by a Medtronic Inc / Royal Academy of Engineering Research Chair [RCSRF1819\7\34].

<ol>
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A., Jayamohan, J., Kuhl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+3D+Printing+to+Create+Personalized+Brain+Models+for+Neurosurgical+Training+and+Preoperative+Planning.">Using 3D Printing to Create Personalized Brain Models for Neurosurgical Training and Preoperative Planning.</a> World Neurosurgery. 90, 668-674 (2016).</li><li>Weinstock, P., 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=Creation+of+a+novel+simulator+for+minimally+invasive+neurosurgery:+Fusion+of+3D+printing+and+special+effects.">Creation of a novel simulator for minimally invasive neurosurgery: Fusion of 3D printing and special effects.</a> Journal of Neurosurgery: Pediatrics. 20 (1), 1-9 (2017).</li><li>Grillo, F. W., 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=Patient-specific+neurosurgical+phantom:+assessment+of+visual+quality,+accuracy,+and+scaling+effects.">Patient-specific neurosurgical phantom: assessment of visual quality, accuracy, and scaling effects.</a> 3D Printing in Medicine. 4 (1), (2018).</li><li>Tsai, A., 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=Creation+and+Validation+of+a+Simulator+for+Neonatal+Brain+Ultrasonography:+A+Pilot+Study.">Creation and Validation of a Simulator for Neonatal Brain Ultrasonography: A Pilot Study.</a> Academic Radiology. 24 (1), 76-83 (2017).</li><li>Reinertsen, I., Collins, D. 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The authors do not have any conflicts of interest to declare.

This protocol details the fabrication process of a patient specific brain phantom, which includes the skull, brain, and vestibular schwannoma tumor. 3D printing methods allowed for anatomically accurate detail to be achieved. The phantom described here was successfully manufactured with the desired level of anatomical detail; CT and ultrasound imaging were used to demonstrate that the tumor was easily visualized with both modalities. The tissue mimicking material, PVA-c, is well established as a tissue-mimicking material for ultrasonic phantoms; its acoustic and mechanical properties can be tuned with additives and the number of freeze-thaw cycles. The material is readily available, simple to use and non-toxic. With repeated use, the phantom had sufficient durability to withstand manipulation and contact with an ultrasound probe during physical simulations of vestibular schwannoma surgery.
Several key steps were identified as being critical to the fabrication process. First, the segmentation of structures for inclusion in the phantom must include the desired level of anatomical detail. The creation of accurate STL files and 3D molds then follows naturally. Secondly, the positioning of planes within the cerebellum mold in step 3.1.9 must be considered carefully, so that the phantom can be readily removed, without damage; it must be cut into enough pieces to allow anatomical details to be retained, whilst enabling the phantom to be removed without getting stuck in the mold. In this case, several iterations were tested and finally the mold was cut into four separate pieces. The third key consideration is that during the PVA-c manufacturing process (section 4), the PVA-c must be left to cool to room temperature (step 4.1.6). If this step is missed and hot PVA-c is added to the molds, it can cause the molds to melt or distort. It is also crucial that once the glass spheres are added (steps 5.1.2 &#8211; 5.1.4), the PVA-c is not left to sit for more than around 10 minutes; if left for a prolonged period of time, the glass spheres will settle to the bottom, and the resulting phantom will have inhomogeneous ultrasound contrast29. Once the glass spheres are added, the PVA-c must be added directly into the molds and placed into the freezer. After the first freeze cycle, the glass spheres will be secured in the place, and the phantom can be used at room temperature. Finally, it is important that the molds are carefully sealed (e.g., with tape) before the PVA-c is added, to minimize leakage of the mixture through gaps where the separate pieced of the mold joined together.
The protocol has several limitations. For example, some specialist equipment is required, including a water bath and an electronic stirrer. A sonicator is also used as part of this protocol, but the sonication step (5.1.3) could be replaced with additional electronic stirring; however, with this alternative, it would take longer to achieve a homogeneous mixture than is possible with the use of sonication. One limitation of PVA-c is that it degrades over time and becomes moldy. The addition of potassium sorbate, as described here, increases the phantom&#8217;s shelf-life, although it must still be kept in an air-tight container. A second limitation of PVA-c is that freeze-thaw cycles are required, which increases the amount of time required to make a phantom. To minimize phantom fabrication time, a key consideration is the speed of freezing and thawing; once the phantom is either fully frozen or fully thawed, the time that it remains in that state does not significantly affect the final phantom16,30. Therefore, the cycle lengths used can be varied, provided that the phantom is fully frozen and thawed at each stage in the cycle. For instance, the tumor in the phantom of this study is very small, so shorter cycles could be used for the tumor than for the brain. Finally, 3D printing the molds and skull is a time-consuming process which consumes a significant portion (3 days) of the total time (1 week) required to fabricate a phantom with this protocol. The printer used was a commercial model from 2018; the printing process could be completed in shorter time frames with the use of newer, faster printers.
The brain phantom presented here could be used directly for clinical training and validation of neuronavigation systems. As the tissue mimicking material, PVA-c enables the resulting phantom to be used repeatedly, for example as a training tool or for the validation of intraoperative ultrasound in vestibular schwannoma surgery, as it is a durable and non-toxic material. As such, the fabrication method is complementary to those previously described in which 3D printing was used to create patient specific brain phantoms20,21,22,23,24,25. The use of PVA-c as the TMM makes the phantom suitable for use in simulation of neurosurgery, as the material can withstand repeated manual manipulation and contact from an ultrasound probe. This work sets the stage for further quantitative validation studies. The phantom method described here is very versatile and could be used to fabricate many types of patient-specific tumor phantoms, extending from the brain to other organs, with compatibility across several imaging modalities.

Phantoms mimicking the specific properties of biological tissues are a useful resource for various experimental and teaching applications. Tissue-mimicking phantoms are essential to characterize medical devices prior to their clinical use1,2 and anatomical phantoms are frequently used in the training of medical staff in all disciplines3,4,5,6,7. Patient-specific anatomical phantoms made with appropriate tissue-mimicking properties are often a critical part of the testing environment and can increase the confidence of clinicians who are learning to use a new device8. However, high manufacturing costs and complex fabrication processes often preclude the routine use of patient-specific phantoms. Here, a method is described for manufacturing a durable, patient-specific brain tumor model using readily available, commercial materials, which can be used for the training and validation of intraoperative ultrasound (US) using computerized tomography (CT) imaging. The phantom described in this study was created using data from a patient with a vestibular schwannoma (a benign brain tumor arising from one of the balance nerves connecting the brain and inner ear) who subsequently underwent surgery and tumor resection via a retrosigmoid suboccipital craniotomy10. The phantom was developed in order to test and validate an integrated intraoperative navigation system for use during this type of brain tumor surgery.
In order to be suitable for this application, the brain tumor phantom needs to possess several key properties. First, it should be made of non-toxic materials, so it can safely be used in a clinical training environment. Second, it should have realistic imaging properties; for the intended application, these specifically include ultrasound attenuation and CT contrast. Third, it should have similar mechanical properties to human tissue so that it can be handled in the same way. Fourth, the phantom should be based on real patient data, so that it is anatomically accurate and can be used for surgical planning and training. Finally, the materials used must be durable, so that the phantom can be used repeatedly.
In general, the tissue-mimicking material and fabrication method chosen for a phantom depends on the intended application. For rigid structures like the skull, the chosen property should not deform or be water-soluble and it should be able to maintain an accurate level of anatomical detail with repeated use; this is especially important when using the phantom for experiments where image registration is used and for surgical simulation purposes. Mineral oil based materials such as gel wax have been promising for ultrasound9,11,12 and photoacoustic13 imaging applications, however, when subjected to repeated mechanical deformation they become friable, so cannot withstand extended use, especially with standard microsurgical neurosurgery instruments. Agar and gelatin are aqueous materials that are also commonly used as tissue-mimicking materials. The additives needed to adjust the acoustic properties of these materials are well known14, but they have limited mechanical strength and are not particularly durable so are not suitable for this application, where the phantom needs to be repeatedly handled.
Polyvinyl alcohol cryogel (PVA-c) is a popular choice of tissue-mimicking material, because its acoustic and mechanical properties can easily be tuned by varying its freeze-thaw cycles. It has been shown that the properties of PVA-c are similar to those of soft tissues15,16,17,18. PVA-c based brain phantoms have been used successfully for ultrasound and CT imaging19. The material is strong enough to be used repeatedly, and it has a high degree of elasticity, so phantom tissue made of PVA-c can be manipulated without being permanently deformed. Polylactic acid (PLA) is a readily available rigid material and was used to manufacture the skull, however, a different printing material can be used in place of PLA, if it has similar mechanical properties and is not water soluble.
Brain phantoms in particular have been fabricated using different methods, depending on the level of complexity required and the tissues that need to be replicated20,21,22,23. Usually, a mold is used, and liquid tissue-mimicking material poured into it. Some studies have used commercial molds24 whilst others use 3D-printed custom molds of a healthy brain, and simulate brain lesions by implanting marker spheres and inflatable catheters19,25. To the best of the author&#8217;s knowledge, this is the first report of a 3D-printed patient-specific brain tumor phantom model created with tissue-mimicking ultrasound and X-ray properties. The total fabrication is visualized by the flowchart in Figure 1; the whole process takes around a week to complete.

<table>
	<tbody>
		<tr>
			<td>AutodeskFusion 360</td>
			<td>Autodesk Inc., San Rafael, California, United States</td>
			<td>https://www.autodesk.co.uk/products/fusion-360/overview</td>
			<td>CAD software</td>
		</tr>
		<tr>
			<td>Barium sulphate</td>
			<td>Source Chemicals</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CT scanner</td>
			<td>Medtronic Inc, Minneapolis, USA</td>
			<td>-</td>
			<td>O-arm 3D mobile X-ray imaging system</td>
		</tr>
		<tr>
			<td>Glass microspheres</td>
			<td>Boud Minerals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical stirrer</td>
			<td>IKA</td>
			<td>4442002</td>
			<td>Eurostar Digital 20, IKA</td>
		</tr>
		<tr>
			<td>Meshmixer</td>
			<td>Autodesk Inc., San Rafael, California, United States</td>
			<td>http://www.meshmixer.com</td>
			<td>3D modelling software. Version 3.5.484 used</td>
		</tr>
		<tr>
			<td>Neuronavigation system</td>
			<td>Medtronic Inc, Minneapolis, USA</td>
			<td>-</td>
			<td>S7 Stealth Station</td>
		</tr>
		<tr>
			<td>PLA</td>
			<td>Ultimaker (Ultimaker BV, Utrecht, Netherlands)</td>
			<td>UM9016</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium sorbate</td>
			<td>Meridianstar</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>PVA</td>
			<td>Ultimaker</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>PVA powder</td>
			<td>Sigma-Aldrich</td>
			<td>363146</td>
			<td>99%+ hydrolysed, average molecular weight 85,000-140,000</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Fisher Scientific</td>
			<td>12893543</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimaker Cura</td>
			<td>Ultimaker BV, Utrecht, Netherlands</td>
			<td>https://ultimaker.com/software/ultimaker-cura</td>
			<td>3D printing software. Version 4.0.0 used</td>
		</tr>
		<tr>
			<td>Ultimaker S5 Printer</td>
			<td>Ultimaker BV, Utrecht, Netherlands</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound scanner</td>
			<td>BK Medical, Luton, UK</td>
			<td>-</td>
			<td>BK 5000 scanner</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>IKA</td>
			<td>20009381</td>
			<td>HBR4 control, IKA</td>
		</tr>
		<tr>
			<td>3D Slicer</td>
			<td>http://slicer.org</td>
			<td>-</td>
			<td>Software used to segment patient data. Version 4.10.2 used</td>
		</tr>
	</tbody>
</table>

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.

Following the described protocol, an anatomically realistic phantom was fabricated, which consists of a patient-specific skull, brain and tumor. The relevant anatomical structures for the phantom (skull, brain, tumor) are segmented using patient MRI and CT data (Figure 2a,b). The patient intra-operative ultrasound data (Figure 2c; Figure 2d shows the same image as Figure 2c, but with the tumor outlined) was used to compare the phantom images to the real patient images.
Meshes were created for each piece of the model (Figure 3), and these were then used to manufacture the 3D molds. The molds were easily printed on a commercial printer and assembled by slotting the pieces together. The cerebellum mold was the most complex to design and assemble (Figure 4). The skull (Figure 5a) was the most difficult part to print as it required support material, so was a slow process; the whole print took a total of three days to complete, which is a limiting factor in the protocol.
The completed phantom (Figure 5) was a realistic model of a patient skull, brain and tumor. The two brain hemispheres (Figure 5b) were produced separately, and have a realistic appearance, featuring the gyri and sulci of the brain. The whole phantom is white in color, as this is the natural color of PVA-c; this can easily be changed by adding dye but was not necessary for the application. The cerebellum (Figure 5c) fits comfortably into the base of the printed skull and the brain hemispheres sit on top of this. The tumor is easily visible in the cerebellum, as the extra contrast added to the tumor results in it being an off-white color that separates it from the surrounding material, which is it securely attached to.
The phantom was imaged with both CT and ultrasound (Figure 6a,b). Barium sulfate was used to give the tumor appropriate CT contrast, and the phantom image (Figure 6a) shows that this was achieved, as the tumor is clearly visualized. The skull was not printed with 100% infill, in order to reduce the time taken for printing. Therefore, the skull does not look entirely realistic in the CT images, because the lattice structure of the print can be seen. This is not a problem for the application, as only the outline of the skull is needed for the neuronavigation system. The skull could be printed with 100% infill to avoid this reduced accuracy of the CT image, but would add time onto the printing process. Glass microspheres were added to the cerebellum, brain hemispheres and tumor for ultrasound contrast. The results show that the tumor is also visible with ultrasound imaging (Figure 6b) and can be distinguished from the surrounding tissue. On visual inspection, the ultrasound images obtained from the phantom (Figure 6b), and those obtained from the patient (Figure 2c) show that the contrast agents used in the phantom were effective for creating realistic imaging properties.
The phantom was tested during surgical simulation in a virtual operating room (Figure 7). The phantom model was positioned on the surgical operating table using a standard skull clamp and the CT scan of the phantom was registered using a clinical neuronavigation system. A retrosigmoid approach to the tumor was simulated and the tumor was imaged using a clinical ultrasound system with a burr hole ultrasound transducer. During the surgical simulation, the phantom model proved to be stable and no damage was observed from manipulating the phantom in the same way the human brain would be during this procedure, so it could be used repeatedly under the same conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61344/61344fig01.jpg" /> 
Figure 1: Flowchart to show the steps required to make a patient specific PVA-c brain phantom. <a href="https://www.jove.com/files/ftp_upload/61344/61344fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61344/61344fig02.jpg" /> 
Figure 2: Patient data used to create phantom model.&#160;Data sources of a patient with a left sided vestibular schwannoma: (a) axial contrast-enhanced T1-weighted MRI, white arrow pointing towards tumor; (b) axial non-contrast CT scan windowed to highlight bone, white arrow pointing towards an expanded internal auditory meatus caused by the tumor; (c) intraoperative ultrasound image obtained during vestibular schwannoma surgery; (d) annotated intraoperative ultrasound image <img alt="Equation 1" src="/files/ftp_upload/61344/icon01.jpg" />: tumor (hyperechoic on ultrasound), <img alt="Equation 4" src="/files/ftp_upload/61344/icon04.jpg" />: brain (cerebellum). <a href="https://www.jove.com/files/ftp_upload/61344/61344fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61344/61344fig03.jpg" /> 
Figure 3: Completed meshes for each section of the phantom.&#160;STL mesh for (a,b) skull, <img alt="Equation 2" src="/files/ftp_upload/61344/icon02.jpg" />: left sided retrosigmoid craniotomy; (c,d) cerebral hemispheres; (e,f) tumor and cerebellum, <img alt="Equation 1" src="/files/ftp_upload/61344/icon01.jpg" />: tumor. <a href="https://www.jove.com/files/ftp_upload/61344/61344fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61344/61344fig04.jpg" /> 
Figure 4: 3D printed cerebellum mold.&#160;3D printed cerebellum mold fully constructed (top left) and the separate pieces, which are numbered from 1 to 4. The hole in piece 2 (denoted by &#8216;H&#8217;) enables the PVA-c to be poured into the mold. <a href="https://www.jove.com/files/ftp_upload/61344/61344fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61344/61344fig05.jpg" /> 
Figure 5: Completed phantom.&#160;The finished phantom (a) skull&#160;(b) phantom with skull top removed: <img alt="Equation 2" src="/files/ftp_upload/61344/icon02.jpg" />: retrosigmoid craniotomy, <img alt="Equation 1" src="/files/ftp_upload/61344/icon01.jpg" />: tumor, <img alt="Equation 4" src="/files/ftp_upload/61344/icon04.jpg" /> brain (cerebellum), <img alt="Equation 5" src="/files/ftp_upload/61344/icon05.jpg" />brain (right cerebral hemisphere); (c) cerebellum and tumor: <img alt="Equation 1" src="/files/ftp_upload/61344/icon01.jpg" />: tumor, <img alt="Equation 4" src="/files/ftp_upload/61344/icon04.jpg" /> brain (cerebellum). <a href="https://www.jove.com/files/ftp_upload/61344/61344fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61344/61344fig06.jpg" /> 
Figure 6: CT and ultrasound images acquired with the phantom.&#160;(a) Axial CT image of complete phantom through the level of the skull base and tumor, (b) Intraoperative ultrasound image of phantom acquired with burr hole ultrasound probe through the retrosigmoid craniotomy in a plane approximately perpendicular to the skull (Simulating surgery, the cerebellum was retracted slightly in order to image directly on the tumor). <img alt="Equation 1" src="/files/ftp_upload/61344/icon01.jpg" />: tumor, <img alt="Equation 4" src="/files/ftp_upload/61344/icon04.jpg" /> brain (cerebellum), <img alt="Equation 2" src="/files/ftp_upload/61344/icon02.jpg" />: left sided retrosigmoid craniotomy. <a href="https://www.jove.com/files/ftp_upload/61344/61344fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61344/61344fig07.jpg" /> 
Figure 7: Testing the phantom during surgical simulation.&#160;Testing the phantom model through surgical simulation in a virtual operating room. <img alt="Equation 6" src="/files/ftp_upload/61344/icon06.jpg" />: neuronavigation system displaying the registered scan of the CT phantom model, <img alt="Equation 3" src="/files/ftp_upload/61344/icon03.jpg" />: ultrasound system used to image the phantom with a burr hole ultrasound transducer (seen positioned next to the ultrasound monitor). Note the model pictured here is based on data acquired from different patient with a right sided tumor. <a href="https://www.jove.com/files/ftp_upload/61344/61344fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning at JoVE.com

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

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 ...

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Research

JoVE Journal

Bioengineering

8.2K Views.  University College London. 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.

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

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

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 ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

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.

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 ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...