Parts of this work have been presented as a poster at the annual meeting of the German Neurosurgical Society (DGNC) 2019 in W&#252;rzburg, Germany and as a short presentation at the annual meeting of the German Society for Computer and Robot Assisted Surgery (CURAC) 2019 in Reutlingen, Germany.

The protocol was approved by the responsible local ethics committee (Ethikkommission der Landes&#228;rztekammer Rheinland-Pfalz, Deutschhausplatz 3, 55116 Mainz, Germany). All institutional guidelines for the care and use of patient data were followed.
1. Segmentation of medical imaging data and creation of a virtual 3D model
NOTE: The software we used for segmentation was Amira 5.4.5. The segmentation process can also be accomplished by using open source software (e.g., 3D Slicer, <a href="https://www.slicer.org/" target="_blank">https://www.slicer.org/</a>)
<ol>
	<li>Use imaging data with high spatial resolution (e.g., a slice thickness of 1 mm or less). Here, a cranial CT dataset with a slice thickness of 0.5 mm and additional MRI data with a slice thickness of 1 mm were used. Use CT data for the segmentation of bone, contrast enhanced T1 MRI images for the segmentation of tumor and neural structures, as well as time-of-flight (TOF) images for vessels.</li>
	<li>Download the DICOM files on the computer and open the segmentation software. Import the files of the different imaging modalities and select the folder with the imaging data.</li>
	<li>Click on the CT images and connect them with a volume rendering module (Volren). Choose Specular for a more realistic rendering and adjust the color transfer slider to visualize bone only. Continue by importing the MRI sequences and connect them to a volume rendering module as well.</li>
	<li>Registration
	<ol>
		<li>Because the MRI and CT images do not overlap, it is necessary to fuse the different imaging data. Therefore, right click on the MRI dataset and choose Compute &#62; Affine Registration. Choose Reference by clicking on the white square of the module, then drag the cursor onto the CT.</li>
		<li>In the Registration Module&#39;s properties leave all settings on default and click on Align Centers, followed by clicking Register. The two different imaging datasets are now fused. Repeat this step for all further imaging datasets.</li>
	</ol>
	</li>
	<li>Verification of matching accuracy:
	<ol>
		<li>Check the matching accuracy by hiding the volume renderings (click on the orange square of the module) and adding an OrthoSlice module to the MR images. Click on the white triangle and choose Colorwash. Next click on the white square, choose Data and connect this port with the CT data by dragging the mouse onto it.</li>
		<li>Adjust the color slider to visualize the neural structures superimposed to the bony skull structures. Check for any misalignments by toggling the weight factor slider while looking at the border between the skull and brain surfaces as well as the ventricles. Repeat this procedure on different slices in coronal and sagittal directions.</li>
	</ol>
	</li>
	<li>Volumetric editing
	<ol>
		<li>Deactivate the OrthoSlice module&#39;s visibility and reactivate volume rendering of the CT. Go to the CT data and look for the lowest value in the dataset, in this case -2,048.</li>
		<li>Next add a Volume Edit module, connect the Volren module with the output data, and set the Padding Value to -2,048.</li>
		<li>Click on Cut Inside and mark the region to be removed in the 3D viewport. 
		NOTE: It is important to avoid overlapping with parts not intended to be removed.</li>
		<li>In this example, parts of the mandible bone and the upper cervical vertebrae were removed.</li>
	</ol>
	</li>
	<li>Segmentation of bone
	<ol>
		<li>Next, the remaining bone must be segmented and converted into a surface mesh. To do so, click on the Segmentation Editor, choose the modified CT image sequence, and add a new Labelset by clicking on New.</li>
		<li>Now choose Threshold as a segmentation option. Set the lower slider to a value of ~250 in case of a CT. Ensure that thin bone structures such as the temporal bone or the upper orbital region are selected in the preview. Otherwise, adjust the lower threshold but avoid selecting any soft tissue.</li>
		<li>Next click on Select and finally add the selection to the labelset (by clicking on the red plus icon).</li>
		<li>Return to the Pool View. A new labelset has been created for the CT. Right click and choose Compute &#62; Surface Gen, check the Compactify option, and click Apply.</li>
		<li>Finally, add a SurfaceView module and adjust the color of the generated mesh.</li>
	</ol>
	</li>
	<li>Segmentation of other structures
	<ol>
		<li>Add other relevant structures by repeating the previous steps. In case of the tumor, manual segmentation was used rather than a thresholding operation.</li>
		<li>To perform manual segmentation, go to the Segmentation Editor, and choose the manual segmentation option (brush icon) to mark structures such as the tumor in every slice. Finally add the selection again by clicking onto the plus icon. Thus, the tumor, the optic nerve, and the intracranial vessels will be segmented and added to the model.</li>
	</ol>
	</li>
	<li>Exporting meshes
	<ol>
		<li>Finally export the generated meshes in the STL format by right clicking on the mesh and clicking on Save. Choose binary STL as the file format.</li>
	</ol>
	</li>
</ol>
2. Preparation of the virtual 3D model for multicolor printing
NOTE: The software used for print preparation in this protocol is Netfabb Premium 2019.0. Autodesk offers free use of this software in its educational program.
<ol>
	<li>Import data and perform automatic repair.</li>
	<li>Open the print preparation program and import the meshes generated in the previous steps as new parts. Check Automatic Repair and click Import.</li>
	<li>Deleting small loose parts
	<ol>
		<li>Select the skull and split its shells into parts by clicking on Modify &#62; Split Shells into Parts. This separates any loose objects not connected to the skull bone.</li>
		<li>Select the skull bone and toggle its visibility off.</li>
		<li>Now select all other parts and delete them.</li>
		<li>Toggle the skull&#39;s visibility on again.</li>
		<li>Repeat this step for all other objects.</li>
	</ol>
	</li>
	<li>Remove overlapping regions. 
	NOTE: In some regions, such as the tumor inside the petrous apex of the skull, the geometries of both objects intersect each other. To avoid printing errors, it is necessary to remove such intersections.
	<ol>
		<li>Select the two intersecting objects and click on Boolean Operations.</li>
		<li>Move the object to be subtracted from the other one to the red side of the list and click on Apply. Now the two objects are clearly separated. This should be checked by toggling their visibility.</li>
		<li>Repeat these steps to allow the tumor as well as the artery inside the tumor to become clearly separated from each other.</li>
	</ol>
	</li>
	<li>Add supporting structures where needed.
	<ol>
		<li>In case of the basilar artery, additional supports are needed to prevent the object from being a loose part after printing.</li>
		<li>Add a new object, in this case a cylinder (File &#62; Part Library), and adjust its dimensions and subdivisions as needed.</li>
		<li>Place the cylinder to completely intersect with the skull and the vessel geometry.</li>
		<li>Now perform the Boolean operation again to subtract the parts within the bone and the blood vessel.</li>
		<li>Repeat this step to add more supports where needed (e.g., the optic nerve).</li>
	</ol>
	</li>
</ol>
3. Preparation for volumetric coloring of selected parts
NOTE: To allow volumetric coloring of certain parts it is necessary to generate not only one surface shell but many subshells (additional surfaces) inside the object.
<ol>
	<li>Select the tumor, in this case, and generate a new shell from it (right click &#62; Modify &#62; Generate Shell).</li>
	<li>Set a shell thickness of 0.3 mm in the Inner Offset Mode with an accuracy of 0.15 mm and Apply. Select the Keep Original Part checkbox. This generates an inner shell with a distance of 0.3 mm to the original surface.</li>
	<li>Select the outside surface of both shells and generate a new shell from it. Select a shell thickness of 0.25 mm in Hollow Mode with an accuracy of 0.15 mm. Also select the Remove Original Part checkbox. This generates a space of 0.05 mm between the two adjacent shells.</li>
	<li>Repeat steps 3.1&#8211;3.3 so that multiple inner shells with constant thicknesses and invariant offsets are created. 
	NOTE: It is recommended to use a shell thickness of 0.35&#8211;0.25 mm as well as an offset of 0.1&#8211;0.05 mm to achieve smooth volumetric coloring.</li>
	<li>Repeat steps 3.1&#8211;3.4 with all other objects, such as the blood vessels.</li>
</ol>
4. Coloring and exporting of the 3D model
NOTE: The coloring of all parts of the model, including the distinct nested shells, is done using the Netfabb software.
<ol>
	<li>Select a part to be colored in the Parts Menu on the left side. Double click the Texture and Color Mesh icon. Choose a color by clicking the color bar on the right side. In the top menu left click on the Paint on Shells icon. Subsequently left click on the model displayed in the screen center. Finally left click on the Apply Changes box in the lower right corner. Be sure to confirm the Remove Old Part selection.</li>
	<li>Repeat these steps with all other objects and shells, respectively.</li>
	<li>Export all objects. Select all objects to be printed, including supports and inner shells, and export them as individual files. Be sure to choose the VRML (WRL) format, because the STL format is not able to transport the color information.</li>
</ol>
5. Printing and post processing of the 3D model
<ol>
	<li>Set up the 3D printer 
	NOTE: The 3DPrint Software (Version 1.03) was used to control the ZPrinter 450 binder jetting machine.
	<ol>
		<li>Open the software and import the colored VRML files by clicking on Open and choosing all relevant data. Click on the Open button in the lower right window corner. In the subsequent window choose millimeters as units. Make sure to check the Keep Position and Orientation as well as the Apply Settings to All Files boxes. Finally choose Z151 as the material type. Click on the Next button.</li>
		<li>For positioning the 3D objects inside of the build volume, mark all objects by pressing the &#60;STRG&#62;+A key.
		<ol>
			<li>In the upper left window, representing the XY view of the build volume, click and drag the marked objects to the center. In the lower left window, representing the XZ view of the build volume, click and drag the objects in the middle of the lower part above the yellow line.</li>
			<li>If a whole skull model is printed, be sure that the opening is facing upwards. If isolated small models are printed, be sure to align delicate parts such as vessels with the XY plane, because this orientation will increase the strength of the respective parts.</li>
			<li>Check the correct orientation of the models by clicking and moving them in the window on the right-hand side.</li>
		</ol>
		</li>
		<li>To prepare for the build process, click on the Setup icon in the upper menu. Make sure that the correct Material Type is selected and that the Layer Thickness is set to 0.1 mm. The Bleed Compensation should be checked and the Print in Monochrome option unchecked.</li>
		<li>To start the printing process, click on the Build icon in the upper menu. In the subsequent window choose Entire Build and click the OK button. Be sure that in the following Printer Status dialogue all listed items are set correctly and that the printer is online. Then, click on the Print button in the lower part of the dialogue box.</li>
	</ol>
	</li>
	<li>Post processing of the model 
	NOTE: Always wear a laboratory coat, gloves, eye protection, and a mask while handling the loose powder and the hardening solution. Always work in a well-ventilated area.
	<ol>
		<li>Unpacking
		<ol>
			<li>After the print is finished, unpack the model by carefully removing the loose powder with the integrated vacuum cleaner. It is important not to directly contact the model with the suction tube to prevent thin structures from breaking apart.</li>
			<li>Remove the model and clean it by applying pressurized air as well as cleaning it with a soft brush. Thicker, more stable, parts of the model can additionally be ground with a harder brush. This optional step allows for a smoother surface finish. Keep in mind that in this state the model is still very fragile.</li>
		</ol>
		</li>
		<li>Infiltration
		<ol>
			<li>Put the model inside a plastic tub.</li>
			<li>Infiltrate it carefully with the hardening solution until no whitish areas are visible.</li>
			<li>The surplus solution has to be removed with pressurized air and disposable paper towels to maintain all the surface details.</li>
			<li>Let the model cure for several hours until it is completely dry.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Frank, G., 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=The+endoscopic+transnasal+transsphenoidal+approach+for+the+treatment+of+cranial+base+chordomas+and+chondrosarcomas.">The endoscopic transnasal transsphenoidal approach for the treatment of cranial base chordomas and chondrosarcomas.</a> Neurosurgery. 59, 50-57 (2006).</li><li>Wu, 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=Quantitative+evaluation+of+different+far+lateral+approaches+to+the+cranio-vertebral+junction+using+the+microscope+and+the+endoscope:+a+cadaveric+study+using+a+tumor+model.">Quantitative evaluation of different far lateral approaches to the cranio-vertebral junction using the microscope and the endoscope: a cadaveric study using a tumor model.</a> Acta Neurochirurgica. 160, 695-705 (2018).</li><li>Huang, X., 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=A+small+3D-printing+model+of+macroadenomas+for+endoscopic+endonasal+surgery.">A small 3D-printing model of macroadenomas for endoscopic endonasal surgery.</a> Pituitary. 22 (1), 46-53 (2018).</li><li>Stone, J. J., Matsumoto, J. M., Morris, J. M., Spinner, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preoperative+Planning+Using+3-Dimensional+Printing+for+Complex+Paraspinal+Schwannoma+Resection:+2-Dimensional+Operative+Video.">Preoperative Planning Using 3-Dimensional Printing for Complex Paraspinal Schwannoma Resection: 2-Dimensional Operative Video.</a> Operative Neurosurgery. 16 (3), 80 (2018).</li><li>Scerrati, 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=A+workflow+to+generate+physical+3D+models+of+cerebral+aneurysms+applying+open+source+freeware+for+CAD+modeling+and+3D+printing.">A workflow to generate physical 3D models of cerebral aneurysms applying open source freeware for CAD modeling and 3D printing.</a> Interdisciplinary Neurosurgery. 17, 1-6 (2019).</li><li>Kamio, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+a+low-cost+desktop+3D+printer+to+develop+a+"one-stop+3D+printing+lab"+oral+and+maxillofacial+surgery+and+dentistry+fields.">Utilizing a low-cost desktop 3D printer to develop a "one-stop 3D printing lab" oral and maxillofacial surgery and dentistry fields.</a> 3D Printing in Medicine. 4, 6 (2018).</li><li>Kondo, K., 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=A+neurosurgical+simulation+of+skull+base+tumors+using+a+3D+printed+rapid+prototyping+model+containing+mesh+structures.">A neurosurgical simulation of skull base tumors using a 3D printed rapid prototyping model containing mesh structures.</a> Acta Neurochirurgica. 158, 1213-1219 (2016).</li><li>Awad, M., Gogos, A. J., Kaye, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skull+base+chondrosarcoma.">Skull base chondrosarcoma.</a> Journal of clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia. 24, 1-5 (2016).</li><li>Jones, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+and+patterns+of+care+in+adult+skull+base+chondrosarcomas+from+the+SEER+database.">Outcomes and patterns of care in adult skull base chondrosarcomas from the SEER database.</a> Journal of Clinical Neuroscience: Official Journal of the Neurosurgical Society of Australasia. 21, 1497-1502 (2014).</li><li>Karakas, A. B., Govsa, F., Ozer, M. A., Eraslan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Brain+Imaging+in+Vascular+Segmentation+of+Cerebral+Venous+Sinuses.">3D Brain Imaging in Vascular Segmentation of Cerebral Venous Sinuses.</a> Journal of Digital Imaging. 32 (2), 314-321 (2018).</li><li>Dong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+brain+arteriovenous+malformation+models+for+clinical+use+and+resident+training.">Three-dimensional brain arteriovenous malformation models for clinical use and resident training.</a> Medicine. 97, 9516 (2018).</li><li>Dolinski, N. D., 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=Solution+Mask+Liquid+Lithography+(SMaLL)+for+One-Step,+Multimaterial+3D+Printing.">Solution Mask Liquid Lithography (SMaLL) for One-Step, Multimaterial 3D Printing.</a> Advanced Materials. 30, 1800364 (2018).</li><li>Coelho, G., 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=Multimaterial+3D+printing+preoperative+planning+for+frontoethmoidal+meningoencephalocele+surgery.">Multimaterial 3D printing preoperative planning for frontoethmoidal meningoencephalocele surgery.</a> Child's Nervous System: ChNS: Official Journal of the International Society for Pediatric Neurosurgery. 34, 749-756 (2018).</li><li>Javan, R., Cho, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Assembled+Prototype+Multimaterial+Three-Dimensional-Printed+Model+of+the+Neck+for+Computed+Tomography-+and+Ultrasound-Guided+Interventional+Procedures.">An Assembled Prototype Multimaterial Three-Dimensional-Printed Model of the Neck for Computed Tomography- and Ultrasound-Guided Interventional Procedures.</a> Journal of Computer Assisted Tomography. 41, 941-948 (2017).</li></ol>

This study was supported by a grant from the medical faculty from the Johannes Gutenberg-University of Mainz to M.K. (&#34;Biomatics - Stufe 1 F&#246;rderung&#34;).

The therapy of intracranial chondrosarcoma is mainly based on complete surgical removal. Often located on the petrous apex, this tumor is close to important structures such as the internal carotid artery, the optic nerve, and the pituitary gland. Therefore, planning the surgical trajectories is a crucial step prior to surgery. Multicolor 3D printing allows for fusion of these structures, each derived from different imaging modalities, into a single object.
During the preparation for 3D printing it is important to carefully select adequate imaging data. High resolution images with a small slice thickness are well suited for 3D reconstruction and smooth transitions, whereas high slice thicknesses will produce coarse, uneven objects. Another critical step of the method is to avoid any intersections of two neighboring objects such as tumor and skull bone. Therefore, Boolean operations have to be performed to subtract one object from the other.
To allow for volumetric coloring it is necessary to create onion shell-like subsurfaces inside an object (Figure 2A,B). It is necessary to have a minimum distance between two adjacent surfaces of at least 0.1 mm to obtain smoothly colored objects (Figure 2D). If the distance chosen is above this value, the individual shells inside the object might become visible (Figure 2C). Attention should be paid to an increased color consumption of the 3D printer when using volumetric coloring. Furthermore, it is also important to check the model for any loose parts and add supports when necessary (e.g., the basilar artery).
The method can only produce stiff, plaster-like material that is not very durable. Especially without the hardening procedure, the model can be easily destroyed during the unpacking procedure. Thus, fragile elements such as blood vessels often tend to break apart.
The technique is also not suitable for simulation of soft tissue. To simulate brain tissue, for example, it might be necessary to either print it with a method that is able to produce soft and hard materials directly12,13 or to print molds that can be used to cast soft objects, such as silicone rubber14. In one test case, the latter method was used to simulate a soft tumor. The limitation of this last procedure was that although the silicone tumor was very flexible, it was necessary to have enough space to insert it into the 3D printed model. Furthermore, it was not possible to create inner structures, such as a blood vessel.
3D binder jetting is an additive manufacturing technique that assembles objects by partial hardening and coloring thin layers of plaster powder. Thus, it allows for printing a nearly unlimited range of colors, color transitions, and colored structures inside the volume of objects in one single process.
Compared to other printing techniques such as filament printers, which produce the lowest costs but only allow two or three colors at once, and Poly Jet printers that produce multicolor, multi- material objects but are very expensive, this technique offers a compromise at an affordable price. The mean material cost for a printed skull was about 150 &#8364;.
With this method it is possible to visualize even more abstract data such as filament fibers derived from MRI fiber tracking sequences or functional imaging depicting, for example, the brain speech area (e.g., Broca&#37;s area).
Apart from surgical simulation, 3D printed, fully colored models of real patient anatomy can help improve the education of medical students or young physicians so they can better understand complex anatomical relations. It is also an important tool in patient education.

Surgical treatment of skull base tumors is a challenging task requiring precise preoperative planning1. Multimodal imaging using computed tomography (CT) and magnetic resonance imaging (MRI) provides the surgeon with information about the patient&#39;s individual anatomy. In clinical practice, this diagnostic information is visualized by displaying a series of two-dimensional (2D) cross-sections representing different aspects of the anatomy (e.g., CT for visualization of bone, CT angiography for vessels, MRI for soft tissue).
However, especially for beginners, medical students, and patients, understanding the complex relations of the different 3D structures to the cross-sectional images is challenging. Beside cadaveric studies2, this problem could be addressed by establishing real-size anatomical models of individual pathologies, displaying anatomical structures in different colors3.
Thanks to technical advances in the last years, 3D printing technologies allow cost-effective construction of complex shapes4,5. Therefore, this technique offers the possibility to construct patient-specific anatomical models that are tangible, clearly depict spatial relations, and can be used for surgical planning and simulation. Especially in rare and complex cases such as petrous apex chondrosarcomas, preoperative simulation of tumor removal in an individual case might help to improve the self-confidence of the surgeon and the patient outcome.
Common FDM-printing (filament deposition modelling) techniques only allow for creating objects with a closed surface in one or a limited variety of colors6. To provide a model for surgical simulation that contains various complex shaped anatomical structures mainly nested inside each other, fully volumetrically colored 3D prints are needed. This allows for a successive removal of tissue layers until an internal structure is revealed.
Plaster-based color 3D binder jetting is a technique able to produce the required multicolor models7. Whereas in its standard configurations only the surface of an object can be colored, herein a modified technique is described to ensure volumetric application of color to internal anatomical structures.
To demonstrate this technique, cases of patients with skull base chondrosarcomas were selected as an example. Chondrosarcomas account for 20% of all neoplasia in the skeletal system, mostly located in the long bones. Primary skull base chondrosarcomas are a rare condition responsible for 0.1&#8211;0.2% of all intracranial tumors8. Mainly located at the petrous apex, these tumors grow in a complex anatomical environment involving pivotal structures such as the internal carotid artery, the optic and other cranial nerves, as well as the pituitary gland. Treatment of these neoplasms is mainly focused on a total surgical resection, because adjuvant therapies alone (e.g., radiation) are not effective enough9.
Due to the complexity and rarity of this tumor entity, preoperative surgical simulation in a 3D printed skull model might help to better visualize and understand the anatomy and to assist the surgeon achieve complete resection. As shown by others10,11 3D printing of patient-specific models improves both residents&#39; and experienced neurosurgeons&#39; understanding of complex neuroanatomy.
However, creating such individualized models from medical imaging data requires skills in image segmentation, 3D modelling, and 3D printing, especially when anatomical structures are to be printed in different colors. This manuscript intends to make fabrication of the described anatomical models more accessible for others by providing a detailed protocol for converting medical imaging data into virtual 3D models and for the fabrication of multicolored 3D objects.
The workflow mainly consists of four parts: 1) segmentation of medical imaging data and creation of a virtual 3D model; 2) preparation of the virtual 3D model for multicolor 3D printing; 3) preparation for volumetric coloring of selected parts; and 4) 3D printing and post processing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>3D Systems (formerly Zcorp)</td>
			<td>x</td>
			<td>Zprinter Z450</td>
		</tr>
		<tr>
			<td>3D printing software</td>
			<td>3D Systems (formerly Zcorp)</td>
			<td>x</td>
			<td>3DPrint Software (Version 1.03)</td>
		</tr>
		<tr>
			<td>Binder solution for cartridge</td>
			<td>4D Concepts GmbH, Gro&#223;-Gerau, Germany</td>
			<td>42-0100-7001</td>
			<td>VisiJet PXL Binder Cartridge clear 1 x ca. 1 Liter</td>
		</tr>
		<tr>
			<td>Infiltration solution</td>
			<td>4D Concepts GmbH, Gro&#223;-Gerau, Germany</td>
			<td>42-0250-1090</td>
			<td>Color-Bond 90, 1 bottle, 454 g</td>
		</tr>
		<tr>
			<td>Modeling Software for 3D print preparation</td>
			<td>Autodesk, San Rafael, CA, USA</td>
			<td>x</td>
			<td>Netfabb Premium (Version 2019.0)</td>
		</tr>
		<tr>
			<td>Print head for binder</td>
			<td>4D Concepts GmbH, Gro&#223;-Gerau, Germany</td>
			<td>42-0150-2010</td>
			<td>HP 11 print head (C4810A)</td>
		</tr>
		<tr>
			<td>Print head for color</td>
			<td>4D Concepts GmbH, Gro&#223;-Gerau, Germany</td>
			<td>42-0150-2011</td>
			<td>HP 57 printhead C 6657 AE Tricolor</td>
		</tr>
		<tr>
			<td>Printing powder</td>
			<td>4D Concepts GmbH, Gro&#223;-Gerau, Germany</td>
			<td>42-0050-2061</td>
			<td>VisiJet PXL Core Eco Drum ca. 14 kg - ca. 11,47 L</td>
		</tr>
		<tr>
			<td>Segmentation software</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>x</td>
			<td>Amira 5.4.5</td>
		</tr>
	</tbody>
</table>

multicolor 3d printing of complex intracranial tumors in neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

Eight patients with chondrosarcoma of the petrous apex were selected for the study and virtual 3D models were created, each containing bone, tumor, vessels, pituitary gland, and optic nerve crossing. Three models underwent multicolored 3D printing using the plaster-based color 3D binder jetting technique (Figure 1A1,A2). Additionally, a single tumor with an internal artery was created (Figure 1B1) to show the benefits of volumetric coloring compared to surface coloring (Figure 1B2,B3).
These models were used to demonstrate the simulation of a surgical approach (e.g., burr hole creation) and tumor resection. This printing technique allowed for combining anatomical structures derived from different imaging modalities into one single object.
The plaster material had bone-like properties and could be easily drilled without melting. Thus, it was feasible to use it to simulate a surgical access route. After the hardening procedure it was stable enough to reproduce even fragile structures such as the intracerebral vessel tree.
The ability to color the entire volume of the model allowed an object&#39;s internal structure, such as the internal carotid artery travelling through the tumor, to be clearly visualized. By removing layers of tumor with the drill, the red artery was gradually revealed during the surgical simulation.
To prove the accuracy of the technique, 3D models were scanned in a computer tomograph. The models created for printing were superimposed to these scans. A deviation mapping was created, and the accuracy was determined in 50 randomly chosen surface points. A mean deviation of 0.021 mm demonstrates the high accordance of the 3D print compared to the original data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60471/60471fig01.jpg" /> 
Figure 1: Volumetric vs. surface colored 3D prints. A1. Exemplary full color 3D print of a patient with chondrosarcoma at the right petrous apex. A2. Detailed view of anatomical structures (arrow = internal carotid artery bifurcation; O = optic nerve chiasm; T = tumor). B1. Blood vessel crossing the tumor volume and cross-sectional level (dotted line). B2. The conventional multicolor printing technique reveals color only at the surface. B3. The modified technique produces volumetrically colored objects suitable for advanced surgical simulation. <a href="https://www.jove.com/files/ftp_upload/60471/60471fig01large.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/60471/60471fig02.jpg" /> 
Figure 2: Workflow for volumetric coloring of 3D prints. A. Virtual 3D model of a tumor with a blood vessel crossing its volume without inner shells. B. A tumor and a blood vessel with multiple inner shells (distance 0.05 mm). C. An example of a high shell distance (1 mm). The single layers of colored and white shells are still visible. D. An example of small shell distance (0.1 mm). The object&#39;s inner volume is completely colored. <a href="https://www.jove.com/files/ftp_upload/60471/60471fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery at JoVE.com

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

multicolor-3d-printing-of-complex-intracranial-tumors-in-neurosurgery

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JoVE Journal

Medicine

7.0K Views.  University Medical Center of the Johannes Gutenberg University Mainz, Germany. The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Video: Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Combining Augmented Reality and 3D Printing to Display Patient Models on a Smartphone

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) printing (3DP) opens new possibilities in clinical applications. Although these technologies have grown exponentially in recent years, their adoption by physicians is still limited, since they require extensive knowledge of engineering and software development. Therefore, the purpose of this protocol is to describe a step-by-step methodology enabling inexperienced users to create a smartphone app, which combines AR and 3DP for the visualization of anatomical 3D models of patients with a 3D-printed reference marker. The protocol describes how to create 3D virtual models of a patient&rsquo;s anatomy derived from 3D medical images. It then explains how to perform positioning of the 3D models with respect to marker references. Also provided are instructions for how to 3D print the required tools and models. Finally, steps to deploy the app are provided. The protocol is based on free and multi-platform software and can be applied to any medical imaging modality or patient. An alternative approach is described to provide automatic registration between a 3D-printed model created from a patient&rsquo;s anatomy and the projected holograms. As an example, a clinical case of a patient suffering from distal leg sarcoma is provided to illustrate the methodology. It is expected that this protocol will accelerate the adoption of AR and 3DP technologies by medical professionals.

Presented here is a method to design an augmented reality smartphone application for the visualization of anatomical three-dimensional models of patients using a 3D-printed reference marker.

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) ...

3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical field using the constantly developing 3D planning and printing capabilities. Computer-assisted design (CAD) and computer assisted manufacturing (CAM) are used to describe the 3D planning and manufacturing of the product. The planning and manufacturing of 3D surgical guides and reconstruction implants is performed almost exclusively by engineers. As technology advances and software interfaces become more user-friendly, it raises a question regarding the possibility of transferring the planning and manufacturing to the clinician. The reasons for such a shift are clear: the surgeon has the idea of what he wants to design, and he also knows what is feasible and could be used in the operating room. It allows him to be prepared for any scenario/unexpected results during the operation and allows the surgeon to be creative and express his new ideas using the CAD software. The purpose of this method is to provide clinicians with the ability to create their own surgical guides and reconstruction implants. In this manuscript, a detailed protocol will provide a simple method for segmentation using segmentation software and implant planning using a 3D design software. Following the segmentation and stl file production using segmentation software, the clinician could create a simple patient specific reconstruction plate or a more complex plate with a cradle for bone graft positioning. Surgical guides can be created for accurate resection, hole preparation for proper reconstruction plate positioning or for bone graft harvesting and re-contouring. A case of lower jaw reconstruction following plate fracture and nonunion healing of a trauma sustained injury is detailed.

This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical ...

Treatment of Facial Deformities using 3D Planning and Printing of Patient-Specific Implants

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve better results, even in the less experienced hand, or continue without it. As technology develops and becomes more user-friendly, we believe it is time to allow the surgeon the option to plan his/her operations and create his/her own patient-specific surgical guides and fixation plates allowing him full control over the process. We present here a protocol for 3D planning of the operation followed by 3D planning and printing of surgical guides and patient-specific fixation implants. During this process we use two commercial computer-assisted design (CAD) software. We also use a fused deposition&#160;modeling printer for the surgical guides and a selective laser sintering printer for the titanium patient-specific fixation implants. The process includes computed tomography (CT) imaging acquisition, 3D segmentation of the skull and facial bones from the CT, 3D planning of the operations, 3D planning of patient-specific fixation implant according to the final position of the bones, 3D planning of surgical guides for performing an accurate osteotomy and preparing the bone for the fixation plates, and 3D printing of the surgical guides and the patient-specific fixation plates. The advantages of the method include full control over the surgery, planned osteotomies and fixation plates, significant reduction in price, reduction in operation duration, superior performance and highly accurate results. Limitations include the need to master the CAD programs.

As technology develops and becomes more user-friendly, planning of operations and patient-specific surgical guides and fixation plates should be performed by the surgeon. We present a protocol for 3D planning of orthognathic skeletal movements and 3D planning and printing of patient-specific fixation plates and surgical guides.

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...