This work is supported by grants from Natural Science Fund of Guangdong Province (No. 2017A030313597) and Southern Medical University (No. LX2016N006, No. KJ20161102).

1. Drawing a two dimensional (2D)-image of a burr hole ring
<ol>
	<li>Open the 2D computer aided design (CAD) software and then create a graphical document.</li>
	<li>Click Draw | Line and draw a reference point with a solid line on the drawing. Click Modify | Offset, and type the specific offset distance in the command line.</li>
	<li>Click on the object and press by the left mouse button to create a solid line. Click Modify | Trim, select the area to be trimmed and click on the extra line.</li>
	<li>Take the inner burr hole ring for example, draw three different views of the inner ring based on the predetermined size in the CAD software. First, draw the front view and modify the graph carefully until it matches the structure expected (Figure 1d).</li>
	<li>Draw the top view by clicking Draw | Line to construct the reference point first and then click on Draw | Circle | Center, Diameter, and input the quantitative value of specific radius of circle or diameter in the command window. Click on the center of the reference point to form a circle (Figure 1f).</li>
	<li>Draw the left view of the inner burr hole ring with the same approach as that of the front view (Figure 1e).</li>
	<li>Click on Dimension | Diameter, and then click on the circumference to mark the diameter of the circle (Figure 1f).</li>
	<li>Click on Dimension | Linear and mark the length and thickness of all associated structures (Figure 1d,e). Click Dimension | Radius to mark the angle of the chamber (Figure 1d).</li>
	<li>Using the same protocol, construct two-dimensional drawings of the outer burr hole ring, and mark the actual size and the labeling (Figure 1a - c).</li>
	<li>Add technical requirements of the production process, including strength, toughness and lack of cracks. Moreover, smoothing of the outer wall is needed.</li>
	<li>Clink on Save to save the 2D-image of the burr hole ring. 
	NOTE: All of these structures mentioned above are in the units of millimeters (mm).</li>
</ol>
2. Construction of a 3D-image of the burr hole ring
<ol>
	<li>Start the 3D drawing software (see the Table of Materials). Select New | Part | Solid and uncheck using the default template. Select part_solid in new file options and click on Ok to create a new interface for setting up a physical part model.</li>
	<li>Click on Part feature in the menu manager on the right and select Create | Solid | Add sheet. In the SOLID drop-down menu, select Rotate | Done. Click on the trace of the preliminary sketch. Select the &#34;front&#34; plane as the sketch plane, then click default under SKET VIEW.</li>
	<li>Select the dotted line on the right toolbar of the window and draw the top section of the part in the two-dimensional sketch. The specific size shall be subject to the two-dimensional drawing. Then click Conform, and select Done in the Protrusion window of protrusion. Click on the Datum plane icon.</li>
	<li>In menu manager, select Create | Solid | Add sheet, and Rotate | Done. Click Bilateral in the properties menu and click Done.</li>
	<li>Click on Front | Forward | Default and then Datum plane | Dotted Line to construct the cross section of the hook of the outer burr hole ring. Then click Conform followed by Done in menu manager. Input &#34;50&#34;&#160;in Angle in indicated direction[45.0000], and then click Done in the Protrusion window and finally, click on the Coloring button. 
	NOTE: The unit of the angle is degree (&#176;).</li>
	<li>Select Redefine in the part feature and click the line structure of the hook. Input the command Section | Define | Sketch.</li>
	<li>Click the Dotted line icon, create two square embossments on the hook section, then input command OK | Done | Coloring.</li>
	<li>Click the Datum axis icon, then input the command Insert a datum | Cross, click the center axis of the line structure, click Angle in datum plane, and then click the &#34;front&#34; plane in the line structure view. Click input value in the offset menu. Input &#34;-45&#34; in &#34;Angle in indicated direction[45.0000]. 
	NOTE: The unit of the angle is degree (&#176;).</li>
	<li>Click on Features | Copy | Mirror. Click on the hook as the object and input command Done select | Done. Click the datum plane to complete the copy. Similarly, the remaining two hooks are copied in this way. Click on Create concentric circle to construct a circle with a radius of 7.23 mm, click the Segmentation of primitives at selected points icon to remove the unnecessary lines of the circle.</li>
	<li>Click the Solid line button in the right toolbar to create a complete outer wall section. Then input the command OK | Done. 
	NOTE: The unit of the radius is millimeter (mm).</li>
	<li>Input &#34;4&#34; in Enter depth, then click on Coloring. Input the command Mirror | Done. Then click on the object and click Done. Click the datum plane to complete the copy.</li>
	<li>Input the command Copy | Mirror | Done, and select two outer walls in different directions, click Done to conform. Click the datum plane to complete the copy.</li>
	<li>Input the command View | Model Settings | Color and appearance | Add. Adjust the RGB color slider and adjust the color to brown to show the graphic details more visually. Then input the command Close | Settings | OK.</li>
	<li>Click the button Eliminating hidden lines, click the Create concentric circle, continue to create an outer edge on the outer wall, click the Segmentation of primitives at selected points button to remove excess lines, and click the Solid line button to connect the newly added outer edge into a complete section. Click Ok.</li>
	<li>Input &#34;0.8&#34; in Inter depth. Click Ok in the Protrusion window. In menu manager, input the command Copy | Mirror | Done. Click the object and click done. Input the command Generate benchmark | Offset. 
	NOTE: The unit of the depth is millimeter (mm).</li>
	<li>Click the Input value in the Offset and enter &#34;0.4&#34; as the Isometric of the specified direction, then click Done. 
	NOTE: The unit of the offset is millimeter (mm).</li>
	<li>Input the command Copy | Mirror | Done, click the outer wall. Input the command Done select | Done. Click Done select and click Done. Click the datum of the image to complete the copy. In this way, the mirror operation of the outer wall and the square embossing is completed respectively.</li>
	<li>Input the command File | Copy, select save format as STL (*stl) in the part type drop-down menu, enter part number and click Ok.</li>
	<li>In the Output STL dialog box, adjust the chord height to 0.006 and the angle control to 0.00001. Input the command Apply | OK.</li>
	<li>Use the same methods as above to build the 3D image of the inner ring.</li>
</ol>
3. Using 3D printer to print the physical model of burr hole ring
<ol>
	<li>Open the model detecting software, input the command Project | Open, choose one STL file in the Open file pop-up dialog box, then click Open. In this software, a warning will appear if defects are detected in this model (Figure 3). If found, repair the model before printing. If there are no defects, click Output.</li>
	<li>After confirming that the outer ring is complete, input the command Part | Export part | as STL | Save. Use the above instructions to detect the defects of the inner ring.</li>
	<li>Following model detection, the printed path needs to be designed. Open the slicing software, click File | Load model file, click on one STL file and click Open to import.</li>
	<li>Click the left mouse button to choose the moving track of the part, adjust the position of parts. On the left side of the screen, set the print speed to 30 mm/s, printing temperature to 210 &#176;C and bed temperature to 80 &#176;C (Figure 4).</li>
	<li>Click Toolpath to SD to save the file in Gcode format to generate printed path (Figure 3).</li>
	<li>Start the 3D printers, click the Preheating button on the main interface, set the preheat temperature of the bed to 80 &#176;C and the nozzle temperature to 210 &#176;C. Click Print when the temperature rises to the preset value, select the target file and click Confirm to start printing.</li>
	<li>The outer ring will be printed first (Figure 5a). After the bottom supporting grid has been constructed, the printing nozzle begins to construct the outer ring vertically layer by layer (Figure 5b - d). This process takes about 13 min.</li>
	<li>After the outer ring is formed, the printer nozzle continues to make the inner ring on the right side (Figure 5c,d), which takes about 8 min.</li>
	<li>Remove both parts from the platform after cooling and being formed (Figure 5e,f).</li>
</ol>
4. Measurement of absolute error
<ol>
	<li>To measure the absolute error, select five printed parts randomly. Measure and record the parameters of each part with Vernier calipers. Choose the measurement accuracy at 0.02 mm.</li>
	<li>Calculate the mean error of each part and the error range of the absolute error (Figure 6a,b).</li>
</ol>

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The authors have nothing to disclose.

These results showed that the software used were practicable to build 3D models of burr hole rings (Figure 1 and Figure 2), and 3D printing can be used to build solid models with designated materials (Figure 4). In terms of the size of the solid model, there was an absolute error from 0 to 0.59 mm determined through the measurement made by Vernier calipers (Figure 6). To some extent, the error is unavoidable since such absolute error comes from many factors, such as the quality of the printing instrument. Industrial printers can have better precision. In addition, when building smaller and more precise parts, the absolute error is more obvious. In general, as shown in Figure 3, the process that constructed the model and further formed the solid model by 3D printing is effective and feasible. Although there is an absolute error, such error can be reduced by improving the quality of the printers and accurately adjusting the printing parameters.
An innovative burr hole ring for DBS was published previously11. In this study, the same model was applied as an example to further demonstrate the systematic process of making the related implants. Currently, in the limited clinical application of 3D printing, model building generally adopts two methods: Firstly, CAD modeling has been used to generate 3D models for further 3D printing operations12. Secondly, imaging data (like in the format of DICOM) has been used to reconstruct the bone structure of patients in three dimensional models according to CT and MRI data. After rendering, the data could be further converted into editable STL files, and then the highly simulated anatomical structure can be produced by 3D printing12,13,14. Similarly, patching or implanting materials that are highly suitable for morphology can be designed according to the anatomical structure of three dimensional reconstruction15,16,17. This method has been applied in cranioplasty. A previous study showed titanium skull patches constructed by 3D printing technology6. Although using 3D printing technology to construct burr hole rings through credible flow visualization in this study in possible, this modeling method has certain limitations in practice.
Being different from the traditional production of burr hole rings, this study proposed to use 3D printing to construct these implantable parts. In fact, traditional products are mostly uniform in size, which does not apply to some patients with skull shape variation and scalp atrophy. The application of 3D printing would potentially provide the implants customized for different patients. Previous studies have proposed and implemented the application of 3D printing to produce skull fragments for skull defect repairment, and has showed its permanent effect6. The efficacy of DBS for functional neurosurgical diseases has been widely recognized (such as Parkinson&#39;s disease, dyskinesia)18,19,20, but the popularity of this treatment is limited, which may be the result of economic burden caused by high consumable costs. Products made by 3D printing have the advantages of high production efficiency, low cost and customization, which makes 3D printing of great potential in the field. The development and application of this technology may provide more patients with an opportunity to receive DBS surgery. However, there are few reports on the use of 3D printing to produce consumables for DBS in the literature.
Moreover, the burr hole ring constructed by 3D printing can have other advantages. This rapid prototyping product can be used for preoperative demonstration, which will better inform patients and their families about the procedure of electrode implantation and enhance doctor-patient communication effectively. Clinicians can carry out preoperative simulation and surgical training through 3D printed products to maximize the simulation of DBS surgery, which will effectively improve their surgical skills. In the surgical treatment of cerebrovascular tumors and cranioplasty, 3D printed products have been applied to surgical training2,5.
This study used carbon fiber, which has good strength and toughness, as the printing material to show the production process of 3D printing. In practice, many factors of implant material should be considered. Firstly, whether the implant has excellent disinfection performance and can keep its properties unchanged under ethylene oxide and hot steam for a long time12. Secondly, implants need to have good biocompatibility and can be placed for a long time without rejectiong by the body. Thirdly, implants need to have excellent mechanical strength, toughness and chemical resistance.
In this study, the construction of a burr hole ring as an example was demonstrated to systematically describe the process from modeling to 3D printing. This is a complete process example. In the future, the use of CAD software, imaging data (e.g., DICOM) and 3D printing to construct the burr hole ring should be encouraged. As mentioned above, 3D reconstruction of DICOM data obtained by imaging can be further converted into STL files that can be used for 3D printing. This is also the mainstream modeling method in clinical scenarios12,13. This method has not been applied in the DBS surgery.

3D printing has been applied in the medical field since the 1980s, especially in surgery for preoperative simulation, anatomical learning and surgical training1. For example, in cerebrovascular operations, preoperative simulation can be conducted by using 3D printed vascular models2. With the development of 3D printing, the texture, temperature, structure and weight of cerebral blood vessels can be simulated to the greatest extent of clinical scenarios. Trainees can perform surgical operations such as cutting and clamping on such models. This training is very important for the surgeons3,4,5. Currently, titanium patches formed by 3D printing have also gradually been applied6, since the skull prostheses developed by 3D printing after imaging and reconstruction are highly conformal. However, the development and application of 3D printing in neurosurgery is still limited.
The burr hole ring, as a part of the lead fixation device, has been widely used in deep brain stimulation (DBS)7,8,9,10. However, current burr hole rings are made by medical device manufacturers according to the unified specifications and dimensions. This standard burr hole ring is not always suitable for all conditions, such as skull malformation and scalp atrophy. It may increase the uncertainties of operation and reduce the acurracy. The emergence of 3D printing makes it possible to develop individualized burr hole rings for patients in clinical scenarios5. At the same time, the burr hole ring, which is not easy to obtain, is not conducive to extensive preoperative demonstration and surgical training1.
To address the problems mentioned above, we proposed to construct a burr hole ring with 3D printing. A previous study in our lab described an innovative burr hole ring for DBS11. In this study, this innovative burr hole ring will be regarded as an excellent example to exhibit the detailed production process. Therefore, the purpose of this study is to provide a modeling process and a detailed technical process of building a solid burr hole ring using 3D printing.

<table><tbody><tr><td>Adobe Photoshop Version 14.0</td><td>Adobe System?US</td><td>_</td><td>Only available with a paid subscription.</td></tr><tr><td>Allcct 3D printer</td><td>Allcct technology co., LTD, WuHan, China</td><td>201807A794124CN</td><td></td></tr><tr><td>Allcct_YinKe_V1.1</td><td>Allcct technology co., LTD, WuHan, China</td><td></td><td>The software is provided by the 3D printer manufacturer and there is no Catalog number associated with it</td></tr><tr><td>AutoCAD 2004</td><td>Autodesk co., LTD?US</td><td>666-12345678</td><td>Software for 2D models</td></tr><tr><td>Carbon Fibre</td><td>Allcct technology co., LTD, WuHan, China</td><td>PLA175&amp;Oslash;5181&amp;Oslash;3&amp;Oslash;B</td><td>The material is provided by the 3D printer manufacturer</td></tr><tr><td>Netfabb Studio Basic 4.9</td><td>Autodesk co., LTD?US</td><td>-</td><td>The software is provided by a 3D printer manufacturer and is open to access</td></tr><tr><td>Pro/E 2001</td><td>Parametric Technology Corporation, PTC, US</td><td>_</td><td>Software for 3D models; Only available with a paid subscription.</td></tr><tr><td>Vernier caliper&amp;nbsp;</td><td>&amp;nbsp;Beijing Blue Light Machinery Electricity Instrument Co,. LTD, China</td><td>GB/T&amp;ensp;1214.1-1996&amp;ensp;</td><td></td></tr></tbody></table>

application of 3d printing in the construction of burr hole ring for deep brain stimulation implants

3D printing has been widely applied in the medical field since the 1980s, especially in surgery, such as preoperative simulation, anatomical learning and surgical training. This raises the possibility of using 3D printing to construct a neurosurgical implant. Our previous works took the construction of the burr hole ring as an example, described the process of using softwares like computer aided design (CAD), Pro/Engineer (Pro/E) and 3D printer to construct physical products. That is, a total of three steps are required, the drawing of 2D-image, the construction of 3D-image of burr hole ring, and using a 3D printer to print the physical model of burr hole ring. This protocol shows that the burr hole ring made of carbon fiber can be rapidly and accurately molded by 3D printing. It indicated that both CAD and Pro/E softwares can be used to construct the burr hole ring via integrating with the clinical imaging data and further applied 3D printing to make the individual consumables.

Three views of 2D images were built through commercial CAD software (see the Table of Materials). In these images, practical size and technical requirements have also been added (Figure 1). Further, three-dimensional data were constructed in (Figure 2) and saved in STL format (Figure 3). As presented in Figure 4, solid parts were built on the platform of the printer. Choosing five groups of these parts, absolute error and error range was calculated (Figure 6a,b). The result showed that, in outer ring, the maximum absolute error and minimum absolute error were found in the outside diameter of the waist and in the thickness of the top respectively. In the inner ring, the maximum absolute error and the minimum absolute error were found in the inside diameter and thickness of the top respectively. The total error range was [0.00, 0.59] (Figure 6a,b).
The STL file is further be converted to Gcode file in the slicing solfware. After that, the Gcode file is transmitted into the 3D printer using an SD card. In the 3D printer, carbon fiber was fed through feeding port. A temperature control unit was used to control the melting of the carbon fiber and the nozzle was used to control the release of printing material and construct the solid model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59560/59560fig1.jpg" /> 
Figure 1: 2D image of burr hole ring. (a-c) 2D views (front view, left view and top view, respectively) of the outer ring. (d-f) 2D views (front view, left view and top, view respectively) of the inner ring. Unit: mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig1large.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/59560/59560fig2.jpg" /> 
Figure 2: 3D image of the burr hole ring. (a-c) 3D views (front view, left view and top view, respectively) of the outer ring. (d-f) 3D views (front view, left view and top view, respectively) of the inner ring. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59560/59560fig3.jpg" /> 
Figure 3: The flowchart for constructing a burr hole ring via 3D printing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59560/59560fig4.jpg" /> 
Figure 4: The process of slicing the burr hole ring by slicing solfware. In the slicing solfware, the STL model was sliced into 0.1 mm thick layers (the black solid arrows). Parameters such as speed and temperature were set (red box) as follows: printing speed at 30 mm/s, printing temperature at 210 &#176;C and bed temperature at 80 &#176;C. Finally, we pressed Save toolpath, and the STL file was converted into Gcode files for 3D printing directly. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59560/59560fig5.jpg" /> 
Figure 5: The example of constructing burr hole ring via 3D printing. (a)The solid arrow on the left indicated the nozzle and the solid arrow on the right side showed the touching buildplate, which was used to host the solid model. (b) The outer ring (the solid arrow) was constructed on the touching buildplate. (c) The inner ring was built on the touching builplate (the solid arrow). (d) The inner ring was built on the right side of the bed (the solid arrow). (e-f) Example of inner ring and the outer ring (the solid arrow) after polishing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59560/59560fig6.jpg" /> 
Figure 6: Measurement of absolute error. (a) Absolute error and error range of outer rings (AE = | MV - SV |; main structures: (1) outer diameter of the top; (2) outer diameter of the waist; (3) thickness of main body; (4) thickness of the top; (5) width of the hook; (6) inner diameter of the top). (b) Absolute error and error range of inner rings (AE = | MV - SV |; main structures: (1) outside diameter of the top; (2) outer diameter of the bottom; (3) inner diameter; (4) total height; (5) thickness of the bottom; (6) thickness of the top. P = part, MV = measured values, SV = standard values, AE = absolute error, ER = error range. Accuracy = 0.02 mm; Unit = mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/59560fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Outer Burr Hole Ring. Please click here to view this file. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/001outer-STL.stl" target="_blank">(Right-click to download.)</a>
Supplemental File 2: Inner Burr Hole Ring. Please click here to view this file. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59560/002inner-STL.stl" target="_blank">(Right-click to download.)</a>

Watch this Scientific Journal Video about Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants at JoVE.com

Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants

Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants.

3D printing has been widely applied in the medical field since the 1980s, especially in surgery, such as preoperative simulation, anatomical learning and ...

application-3d-printing-construction-burr-hole-ring-for-deep-brain

Nanyang Technological University

Research

JoVE Journal

Behavior

6.8K Views.  Southern Medical University. Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants.

Video: Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants

4D Printed Bifurcated Stents with Kirigami-Inspired Structures

Branched vessels, typically in the form of the letter &#34;Y,&#34;&#160;can be narrowed or blocked, resulting in serious health problems. Bifurcated stents, which are hollow in the interior and exteriorly shaped to the branched vessels, surgically inserted inside the branched vessels, act as a supporting structure so that bodily fluids can freely travel through the interior of the stents without being obstructed by the narrowed or blocked vessels. For a bifurcated stent to be deployed at the target site, it needs to be injected inside the vessel and travel within the vessel to reach the target site. The diameter of the vessel is much smaller than the bounding sphere of the bifurcated stent; thus, a technique is required so that the bifurcated stent remains small enough to travel through the vessel and expands at the targeted branched vessel. These two conflicting conditions, that is, small enough to pass through and large enough to structurally support narrowed passages, are extremely difficult to satisfy simultaneously. We use two techniques to fulfill the above requirements. First, on the material side, a shape memory polymer (SMP) is used to self-initiate shape changes from small to large, that is, being small when inserted and becoming large at the target site. Second, on the design side, a kirigami pattern is used to fold the branching tubes into a single tube with a smaller diameter. The presented techniques can be used to engineer structures that can be compacted during transportation and return to their functionally adept shape when activated. Although our work is targeted on medical stents, biocompatibility issues need to be solved before actual clinical use.

Using a 3D printer, a shape memory polymer filament is extruded to form a branched tubular structure. The structure is patterned and shaped such that it can contract into a compact form once folded and then return to its formed shape when heated.

Branched vessels, typically in the form of the letter &#34;Y,&#34;&#160;can be narrowed or blocked, resulting in serious health problems. Bifurcated ...

Engineering

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Neuroscience

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Bioengineering

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Automated 3D Brain and Skull Modeling from MRI for NHP Neurosurgical Planning

A Neural Implant Design Toolbox for Nonhuman Primates

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) neurosurgical planning. This automated, computational software-based technique provides an efficient way of extracting brain and skull features from MRI files as opposed to traditional manual extraction techniques using imaging software. Furthermore, the procedure provides a method for visualizing the brain and craniotomized skull together for intuitive, virtual surgical planning. This generates a drastic reduction in time and resources from those required by past work, which relied on iterative 3D printing. The skull modeling process creates a footprint that is exported into modeling software to design custom-fit cranial chambers and headposts for surgical implantation. Custom-fit surgical implants minimize gaps between the implant and the skull that could introduce complications, including infection or decreased stability. By implementing these pre-surgical steps, surgical and experimental complications are reduced. These techniques can be adapted for other surgical processes, facilitating more efficient and effective experimental planning for researchers and, potentially, neurosurgeons.

Author Spotlight: Streamlined Brain and Skull Modeling for Enhanced Neurosurgical Planning in NHP Research

This paper outlines automated processes for nonhuman primate neurosurgical planning based on magnetic resonance imaging (MRI) scans. These techniques use procedural steps in programming and design platforms to support customized implant design for NHPs. The validity of each component can then be confirmed using three-dimensional (3D) printed life-size anatomical models.

This paper describes an in-house method of 3D brain and skull modeling from magnetic resonance imaging (MRI) tailored for nonhuman primate (NHP) ...

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.

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. ...

Medicine

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 ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...