We thank AB Nexus and the State of Colorado for their generous support of our scientific research into voxel printing for presurgical planning. We thank L. Browne, N. Stence, and S. Sheridan for providing data sets used in this study. This study was funded by the AB Nexus Grant and the State of Colorado Advanced Industries Grant.

NOTE: 3D Slicer Medical Image Computing Software4 (see the Table of Materials) was used for the work completed in sections 1 through 3.
1. Data input
<ol>
	<li>Open the medical image computing software, click the File button and DICOM from the dropdown menu, and wait for the DICOM Browser window to open.
	<ol>
		<li>In the DICOM Browser window, select Import. Wait for the Import DICOM Files from directory popup window to appear.</li>
		<li>Navigate to the DICOM file stack and click the Import button.</li>
		<li>Ensure that the selected stack of DICOM files are loaded into the DICOM Browser. Ensure that the data has been correctly populated and matches the desired study in the following categories: Patient, Study, Series, and Instance.
		<ol>
			<li>Click the Advanced check box to activate additional metadata. Select the desired Series Number and click the Examine button. Ensure the desired sequence is not displaying warnings. Click the check box next to the desired DICOM Data file | Load. 
			NOTE: Select the highest resolution images with the thinnest slice acquisition as this method is capable of printing at 15 &#181;m and 27 &#181;m slice thickness.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For volume rendering, once the sequence is loaded into the medical image computing software, navigate to Modules and select Volume Rendering Module from the dropdown menu.
	<ol>
		<li>In the Volume Rendering module, select the name of the sequence from the Volume dropdown menu to activate the image stack and translate the data into a voxelized volume. Ensure that the active module&#39;s name matches the desired sequence selected in step 1.1.3.1.</li>
		<li>Click the Eye Ball icon next to the Volume dropdown to visualize the selected volume in 3D. Ensure that the 3D display window is open and a grayscale 3D representation is visible.</li>
		<li>Next, click the arrow next to Advanced to open the Advanced Tools. Select the Volume Property tab to open a set of controls for modifying the color channel of the voxel model.</li>
		<li>Navigate to the Scalar Opacity Mapping menu. Left-click in the field to create points where intensity values will be defined by opacity. Place points along this scale to visualize the anatomy of interest. 
		NOTE: The right-left location of the point is correlated to the range of the image&#39;s intensity values, and the up-down location refers to the opacity.</li>
		<li>Navigate to the Scalar Color Mapping menu. Left-click in the field to create points and specify colors correlated to intensity values. Double-click in the field to open a Select Color window to modify color information.</li>
	</ol>
	</li>
</ol>
2. Manipulations
NOTE: A masking step is required if the anatomy is sufficiently complex, to the point where surrounding tissues and extraneous data are present after modifications to the Volume Properties.
<ol>
	<li>Navigate to Modules and select the Segment Editor from the dropdown menu. Ensure the Segment Editor toolbars appear.
	<ol>
		<li>Navigate to the Segmentation dropdown and select Create New Segmentation as. Type a custom name for the segmentation from the Rename Segmentation popup window and click OK.</li>
		<li>Navigate to the Master Volume dropdown and select the active volume, which will have the same name as the Volume Rendering. Next, click the Add button directly below the dropdown. Ensure the segment container is created in the field below.</li>
		<li>Navigate to the effects tool panel below and select the Scissors tool. Navigate to the Scissors menu and select Fill Inside, Free-form, and Unlimited. Next, hover over the 3D Window, right-click and hold while drawing around the area to be erased. Ensure a colored swath appears, showing what has been covered. Repeat this process until all areas to be deleted are covered. 
		NOTE: There are Extensions, such as Segment Editor Extra Effects, that can be downloaded into the medical image computing software, containing tools for creating this segmentation.</li>
		<li>Next, select the Mask Volume tool from the Effects menu. Check Select Inside to delete all image data covered by the segment. Next, modify the Fill Value to be -1000, which is equal to air, or void, in the Hounsfield unit scale. Finally, hit apply and click the Eye Ball next to the Output Volume to show the new masked volume.
		<ol>
			<li>Navigate to Modules and select Volume Rendering from the dropdown menu. Click the Eye Ball next to the active volume to turn off the visualization.</li>
			<li>Next, from the dropdown menu, select the newly created masked volume. Click the Eye Ball to activate the volume.</li>
			<li>Finally, navigate to the Inputs menu and open the Properties dropdown menu. Select the Volume Property created in step 1.2.5. Ensure the volume in the 3D View is masked and color-encoded.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Slicing
NOTE: This process bypasses the traditional 3D printing method by sending the slice files directly to the 3D printing instead of an STL mesh file. In the following steps, slices will be created from the volume rendering. The Bitmap Generator module is a custom-built extension. This can be downloaded from Extensions Manager.
<ol>
	<li>Navigate to the Modules, select Slicerfab from the dropdown. Ensure the Print Parameters and Output Parameters menus are present.
	<ol>
		<li>Under the Printer Parameters dropdown, ensure the X resolution is set to 600 DPI and the Y resolution is set to 300 DPI. Ensure the Layer thickness is set to 27 &#181;m.</li>
		<li>Next, open the Output Parameters menu and modify the scale of the final model as needed.</li>
		<li>Finally, select a file location for the slices to be saved and click Generate. 
		&#8203;NOTE: This step can take several minutes to complete.</li>
	</ol>
	</li>
</ol>
4. Dithering
NOTE: Adobe Photoshop (see the Table of Materials) was used for the work completed in section 4.
<ol>
	<li>Open the image editing software and click File and select Open from the dropdown menu. Navigate to the first image of the PNG file stack created in the previous step and click the Open button.</li>
	<li>Navigate to Window and select Actions from the dropdown menu. In the Actions menu, click New Action, enter a custom name, and select OK. Ensure the action is being recorded by checking that the Record button is active and red.
	<ol>
		<li>Once the image has loaded, navigate to Image | Mode | Indexed Color. In the Index window, select from the dropdown menu Local Perceptual and specify the number of colors to be 8.</li>
		<li>In the Forced menu, select Custom. Click the first two squares, wait for the Custom Color window to pop up, and select a custom color pallet. Select 100% Magenta and ensure C, Y, and K are set to 0.
		<ol>
			<li>Repeat this process and ensure there are two squares devoted to 100% C, Y, and K.</li>
		</ol>
		</li>
		<li>In the Options menu, for Matte, select Custom from the dropdown menu. For Dither, select Diffusion, and for Amount, select 100%. Finally, click OK.</li>
		<li>Navigate to the Action menu and click the square button to stop recording. Close the active window and click No in the save changes popup window.</li>
	</ol>
	</li>
	<li>Navigate to File | Automate | Batch. In the Batch popup window, navigate to the Action dropdown and select the action created in the previous step. Next, under the Source menu, click the Choose button and navigate to the folder of images exported in step 3.1.3. Under the Destination menu, click the Choose button, select a destination folder location for the new files, and click OK.</li>
</ol>
5. Voxel printing
NOTE: Stratasys GrabCAD5 was used for the work completed in section 5.
<ol>
	<li>Open the print software, click Apps and Launch Voxel Print Utility from the dropdown menu.
	<ol>
		<li>In the Slice Files&#39; Prefix text box, enter the prefix of the PNG file stack. Next, click the Select button and navigate to the folder where the PNG files stack is located, and click OK.</li>
		<li>Under Slice Range, ensure the First Slice and Number of Slices match the number of files in the created folder.</li>
		<li>Under Slicing Parameters, ensure the Sliced thickness (mm) matches the settings specified in step 3.1.1.1 and Slice width (pixels) and Slice height (pixels) match the PNG files width and height.</li>
		<li>Under Background Color, ensure the background matches the background color, set not to print. Once completed, click the Next button.</li>
	</ol>
	</li>
	<li>On the Tools page under Material Mapping, select the material from the dropdown menu to be mapped to the associated color, derived from the PNG files. Repeat this process for each color in the menu. Then, click Finish | OK on the popup window Info Gcvf creation succeeded.</li>
	<li>On the host computer print software, click File | Import File from the dropdown menu. Navigate to the Gcvf file and click Load. On the main screen, select Print.</li>
</ol>

<ol>
	<li>Ali, 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=Clinical+situations+for+which+3D+printing+is+considered+an+appropriate+representation+or+extension+of+data+contained+in+a+medical+imaging+examination:+adult+cardiac+conditions.">Clinical situations for which 3D printing is considered an appropriate representation or extension of data contained in a medical imaging examination: adult cardiac conditions.</a> 3D Printing in Medicine. 6 (1), 24 (2020).</li><li>Ballard, D. H., 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=Radiological+Society+of+North+America+(RSNA)+3D+Printing+Special+Interest+Group+(SIG)+clinical+situations+for+which+3D+printing+is+considered+an+appropriate+representation+or+extension+of+data+contained+in+a+medical+imaging+examination:+abdominal,+hepatobiliary,+and+gastrointestinal+conditions.">Radiological Society of North America (RSNA) 3D Printing Special Interest Group (SIG) clinical situations for which 3D printing is considered an appropriate representation or extension of data contained in a medical imaging examination: abdominal, hepatobiliary, and gastrointestinal conditions.</a> 3D Printing in Medicine. 6 (1), 13 (2020).</li><li>Corney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+and+last+industrial+revolution.">The next and last industrial revolution.</a> Assembly Automation. 25 (4), (2005).</li><li>Fedorov, 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=3D+Slicer+as+an+image+computing+platform+for+the+quantitative+imaging+network.">3D Slicer as an image computing platform for the quantitative imaging network.</a> Magnetic Resonance Imaging. 30 (9), 1323-1341 (2012).</li><li>Guide to Voxel Printing. GrabCAD Available from: <a target="_blank" href="https://help.grabcad.com/article/230-guide-to-voxel-printing?locale=en">https://help.grabcad.com/article/230-guide-to-voxel-printing?locale=en</a> (2021)</li><li>Bader, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+data+matter:+Voxel+printing+for+the+digital+fabrication+of+data+across+scales+and+domains.">Making data matter: Voxel printing for the digital fabrication of data across scales and domains.</a> Science Advances. 4 (5), (2018).</li><li>Zhang, F., Li, C., Wang, Z., Zhang, J., Wang, Y. <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+for+arbitrary+distribution+with+nanoscale+resolution.">Multimaterial 3D printing for arbitrary distribution with nanoscale resolution.</a> Nanomaterials. 9 (8), 1108 (2019).</li><li>Robson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STL+Algorithms.">The STL Algorithms.</a> Using the STL. , 47-54 (1998).</li><li>Waran, V., Narayanan, V., Karuppiah, R., Owen, S. L. F., Aziz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+multimaterial+3D+printers+in+creating+models+with+pathological+entities+to+enhance+the+training+experience+of+neurosurgeons.">Utility of multimaterial 3D printers in creating models with pathological entities to enhance the training experience of neurosurgeons.</a> Journal of Neurosurgery. 120 (2), 489-492 (2014).</li><li>Cumbler, E., 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=Contingency+planning+for+healthcare+worker+masks+in+case+of+medical+supply+chain+failure:+Lessons+learned+in+novel+mask+manufacturing+from+COVID-19+pandemic.">Contingency planning for healthcare worker masks in case of medical supply chain failure: Lessons learned in novel mask manufacturing from COVID-19 pandemic.</a> American Journal of Infection Control. 49 (10), 1215-1220 (2021).</li></ol>

N.J. is an author on a patent application filed by the University of Colorado Regents that describes methods like those described in this work (application no. US16/375,132; publication no. US20200316868A1; filed 04 April 2019; published 08 October 2020). All other authors declare that they have no competing interests.

The current representational framework that the majority, if not all, of digital modeling tools employ today results in the STL file format8. Nevertheless, the specific nature of this paradigm has proven inadequate when trying to express the granular or hierarchical structure of more complex, natural materials. With the arrival of recent additive manufacturing techniques such as multimaterial 3D printing, highly tuned and highly optimized objects can be produced, which display gradual material tra...

Currently, surgeons study numerous discrete 2-dimensional (2D) imaging modalities displaying distinct data to plan for operations on 3D patients. Furthermore, viewing this data on a 2D screen is not fully capable of communicating the full extent of the collected data. As the number of imaging modalities grows, the ability to synthesize more data from distinct modalities, which exhibit multiple scales of organization, necessitates new forms of digital and physical representation to condense and curate information for more effective and efficient surgical planning.
3D-printed, patient-specific models have emerged as a new diagnostic tool for surgical planning that has been shown to reduce operating time and surgical complications1. However, the process is time-consuming due to the standard stereolithography (STL) method of 3D printing, which shows a visible loss of data and renders printed objects as solid, homogeneous, and isotropic materials. As a result, 3D printing for surgical planning has been limited to bony structures and simple morphological descriptions of complex organs2. This limitation is a result of an outdated manufacturing paradigm guided by the products and needs of the industrial revolution, where manufactured objects are fully described by their exterior boundaries3. However, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation that represent the variations across the entire volume, which change point by point.
To address this issue, a 3D visualization and modeling technique (Figure 1) was developed and coupled with a novel, additive manufacturing process that enables greater control over the mixing and deposition of resins in ultrahigh resolution. This method, called bitmap printing, replicates human anatomy by 3D printing directly from medical images at a level of spatial fidelity and spatial/contrast resolution of advanced imaging technology approaching 15 &#181;m. This enables the precise and graduated control required to replicate variations in morphologically complex soft tissue with no loss or alteration of data from diagnostic source images.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Slicer Image Computing Platform</td>
			<td>Slicer.org</td>
			<td>Version 4.10.2&#8211;4.11.2</td>
			<td></td>
		</tr>
		<tr>
			<td>GrabCAD</td>
			<td>Stratasys</td>
			<td>1.35</td>
			<td></td>
		</tr>
		<tr>
			<td>J750 Polyjet 3D Printer</td>
			<td>Stratasys</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop</td>
			<td>Adobe</td>
			<td>2021</td>
			<td></td>
		</tr>
	</tbody>
</table>

voxel printing anatomy: design and fabrication of realistic, presurgical planning models through bitmap printing

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of complex organs due to the fundamental limitations in accuracy, quality, and efficiency of the current modeling paradigm. This has largely ignored the soft tissue critical to most surgical specialties where the interior of an object matters and anatomical boundaries transition gradually. Therefore, the needs of the biomedical industry to replicate human tissue, which displays multiple scales of organization and varying material distributions, necessitate new forms of representation. 
Presented here is a novel technique to create 3D models directly from medical images, which are superior in spatial and contrast resolution to current 3D modeling methods and contain previously unachievable spatial fidelity and soft tissue differentiation. Also presented are empirical measurements of novel, additively manufactured composites that span the gamut of material stiffnesses seen in soft biological tissues from MRI and CT. These unique volumetric design and printing methods allow for deterministic and continuous adjustment of material stiffness and color. This capability enables an entirely new application of additive manufacturing to presurgical planning: mechanical realism. As a natural complement to existing models that provide appearance matching, these new models also allow medical professionals to "feel" the spatially varying material properties of a tissue simulant-a critical addition to a field in which tactile sensation plays a key role.

A positive result, as shown in Figure 2 and Figure 3, will be a direct translation of the volume rendering as defined in steps 1.2.5 or 2.1.1.4. The final model should visually match the volume rendering in size, shape, and color. Along this process, there are numerous steps where an error can occur, which will affect one or more of the properties listed above.
Issues related to the uniform scaling, as shown in <stron...

Watch this Scientific Journal Video about Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing at JoVE.com

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing

This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data.

Most applications of 3-dimensional (3D) printing for presurgical planning have been limited to bony structures and simple morphological descriptions of ...

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2.6K Views.  University of Colorado-Anschutz. This method demonstrates a voxel-based 3D printing workflow, which prints directly from medical images with exact spatial fidelity and spatial/contrast resolution. This enables the precise, graduated control of material distributions through morphologically complex, graduated materials correlated to radiodensity without loss or alteration of data. 

Video: Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Methods Article

Voxel Printing Anatomy: Design and Fabrication of Realistic, Presurgical Planning Models through Bitmap Printing