This work was supported by the Institute of Information &#38; Communications Technology Planning and Evaluation (IITP) grant funded by the Korean government (MSIT) (No. 2018-0-01290, the Development of an open dataset and cognitive processing technology for the recognition of features derived from unstructured humans (police officers, traffic safety officers, pedestrians, etc.) motions used in self-driving cars) and the GIST Research Institute (GRI) grant funded by the GIST in 2019.

1. Blood vessel mock-up design for the demonstration
<ol>
	<li>Set the diameter of the proximal main vessel to 25 mm, the diameters of the distal main vessel and the side branch equal to 22 mm. Set the total length of the vessels equal to 140 mm. Set the length of the proximal main vessel, the distal main vessel and the side branch to 65 mm, 75 mm and 65 mm, respectively. The complete blood vessel is shown in Figure 2 and Figure 3.</li>
	<li>Print the computer model of the branched vessel by using an FDM 3D printer. Use a polycarbonate filament.</li>
</ol>
2. Blood vessel mock-up fabrication by molding
<ol>
	<li>Create a box-shaped container that will house the 3D printed part. Set the container dimensions to 110 x 105 x 70 mm and use an acrylic plate.</li>
	<li>With the 3D printed branched vessel placed at the center of the box, gently pour the silicone inside the container to minimize bubble formation. Dry the liquid silicone and harden it for 36~48 h.</li>
	<li>Remove solidified silicone from the container and cut it in half to remove the 3D printed part. Rejoin the divided silicone at the cut plane. The resulting joined body is the blood vessel mockup. The final result is shown Figure 4.</li>
</ol>
3. Design of the branched stent based on kirigami
NOTE: The size of the branched stent is made to snuggly fit inside the Y-shaped pathway of the blood vessel mockup. The interior is made hollow, and the surface tubular meshes are designed to functionally fold and return to the full unfolded configuration.
<ol>
	<li>Design the trunk of the bifurcated stent following wavy patterns similar to conventional stents. Set the diameter of the trunk to 22 mm and the length of the trunk to 38 mm.</li>
	<li>Design the bifurcated branches to be a cylinder, as shown in Figure 5B. Set the diameter of the branch to 18 mm and the length of the branch to 34 mm.</li>
	<li>Set the total length of the stent to 72 mm. The final shape is shown in Figure 6.</li>
</ol>
4. 3D printing with SMP filaments
<ol>
	<li>Print the bifurcated stent in an FDM 3D printer using an SMP filament. The major composition of this filament is polyurethane. The commercial vendor also provides these filaments in the form of pellets so that the end user can also add additional substances to tailor the characteristics of the material (Figure 7).</li>
	<li>Use slicing software for model slicing and to control the settings of the 3D printer. Set the extruder temperature to 230 &#176;C and the temperature of the printer bed to room temperature. Set the layer height to 0.1 mm to minimize the staircase effect.</li>
	<li>Set the printing speed to 3,600 mm/min. Set the amount of interior fill percentage to 80%. Include the supporter formation during printing, which is needed because the structure is hollow in the interior. Figure 8 illustrates the printing process.</li>
</ol>
5. Smoothing out the surface
NOTE: The following steps are required because rough surfaces can damage the vessels by abrasion.
<ol>
	<li>Remove the supporters using cutters (Figure 9A). The supporters are attached at the interior of the stent. When removing the stents, exercise extreme caution to avoid tearing the stents.</li>
	<li>Rub the surface against sandpaper (Figure 9B) to remove the layer lines, striations, or blemishes on the printed surface. Repeated polishing may be needed where the supporters are removed by the cutters.</li>
	<li>Paint the surface using a spray in a well-ventilated location, and wear a personal mask. Clean, sand, and dry the surface. Protect from overspraying by applying thin layers of repeated paints. Use black paints to enhance the contrast between the silicone vessel mockup and the stent (Figure 9C).</li>
</ol>
6. Deforming the bifurcated stent
<ol>
	<li>Place the bifurcated stents in warm water such that the temperature is above the glass transition temperature. When the stent becomes softened, push one half of the branch against the other half. Nest one half within the other half, as shown in Figure 10A.</li>
	<li>Fold the two branches into a single cylinder so that it can travel through the main vessel. Perform the same nesting process to the other branch. Subsequently, the two halves of the cylinders are closed into one, as shown in Figure 10B.</li>
</ol>
7. Insertion of the bifurcated stent into the vessels
<ol>
	<li>Fill a tank with warm water. Set the water temperature to 55-60 &#176;C. Immerse the silicone vessel mockup inside the tank. Orient the mockup such that the main vessel is above and the branches are below.</li>
	<li>Insert the folded bifurcated stent into the opening of the silicone vessel mockup from above. Orient the folded bifurcated stent such that its branches are towards the opening. The folded bifurcated stent will start to expand, and the lower branches will divide such that each branch will slide towards its mating pathway from the bifurcation core of the Y-shaped vessels (Figure 12).</li>
</ol>

<ol><li>Migliavacca, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stainless+and+shape+memory+alloy+coronary+stents%3A+a+computational+study+on+the+interaction+with+the+vascular+wall%5BTitle%5D)+AND+%22Biomechanics+and+Modeling+in+Mechanobiology%22%5BJournal%5D)">Stainless and shape memory alloy coronary stents: a computational study on the interaction with the vascular wall.</a> Biomechanics and Modeling in Mechanobiology. 2 (4), 205-217 (2004).</li>
<li>Kuribayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-deployable+origami+stent+grafts+as+a+biomedical+application+of+Ni-rich+TiNi+shape+memory+alloy+foil%5BTitle%5D)+AND+%22Material+Science+and+Engineering+A%22%5BJournal%5D)">Self-deployable origami stent grafts as a biomedical application of Ni-rich TiNi shape memory alloy foil.</a> Material Science and Engineering A. 419, 131-137 (2006).</li>
<li>Suwaidi, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immediate+and+long-term+outcome+of+intracoronary+stent+implantation+for+true+bifurcation+lesions%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Cardiology%22%5BJournal%5D)">Immediate and long-term outcome of intracoronary stent implantation for true bifurcation lesions.</a> Journal of the American College of Cardiology. 35 (4), 929-936 (2000).</li>
<li>Mao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+self-folding+structures+by+3D+printed+digital+shape+memory+polymers%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Sequential self-folding structures by 3D printed digital shape memory polymers.</a> Scientific Reports. 5, 13616(2015).</li>
<li>Ge, Q., Qi, H. J., Dunn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+materials+by+four-dimension+printing%5BTitle%5D)+AND+%22Applied+Physics+Letters%22%5BJournal%5D)">Active materials by four-dimension printing.</a> Applied Physics Letters. 103, 131901(2013).</li>
<li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-shape+active+composites+by+3D+printing+of+digital+shape+memory+polymers%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Multi-shape active composites by 3D printing of digital shape memory polymers.</a> Scientific Reports. 6, 24224(2016).</li>
<li>Chen, S., Zhang, Q., Feng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+printing+of+tunable+shape+memory+polymer+blends%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry+C%22%5BJournal%5D)">3D printing of tunable shape memory polymer blends.</a> Journal of Materials Chemistry C. 5, 8361-8365 (2017).</li>
<li>Qi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimaterial+4D+printing+with+tailorable+shape+memory+polymers%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Multimaterial 4D printing with tailorable shape memory polymers.</a> Scientific Reports. 6, 31110(2016).</li>
<li>Lendlein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biodegradable%2C+elastic+shape-memory+polymers+for+potential+biomedical+applications%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Biodegradable, elastic shape-memory polymers for potential biomedical applications.</a> Science. 296, 1673-1676 (2002).</li>
<li>Lendlein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shape+memory+polymers%5BTitle%5D)+AND+%22Angewandte+chemie%22%5BJournal%5D)">Shape memory polymers.</a> Angewandte chemie. 41, 2034-2057 (2002).</li>
<li>Baer, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+and+in+vitro+deployment+of+a+laser-activated+shape+memory+polymer+vascular+stent%5BTitle%5D)+AND+%22BioMedical+Engineering+OnLine%22%5BJournal%5D)">Fabrication and in vitro deployment of a laser-activated shape memory polymer vascular stent.</a> BioMedical Engineering OnLine. 6, 43(2007).</li>
<li>Wache, H. M., Tartakowska, D. J., Hentrich, A., Wagner, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+polymer+stent+with+shape+memory+effect+as+a+drug+delivery+system%5BTitle%5D)+AND+%22Journal+of+Materials+Science%3A+Materials+in+Medicine%22%5BJournal%5D)">Development of a polymer stent with shape memory effect as a drug delivery system.</a> Journal of Materials Science: Materials in Medicine. 14 (2), 109-112 (2003).</li>
<li>Shyu, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+kirigami+approach+to+engineering+elasticity+in+nanocomposites+through+patterned+defects%5BTitle%5D)+AND+%22Nature+Materials%22%5BJournal%5D)">A kirigami approach to engineering elasticity in nanocomposites through patterned defects.</a> Nature Materials. 14, 785-789 (2015).</li>
<li>Rossiter, J., Sareh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kirigami+design+and+fabrication+for+biomimetic+robotics%5BTitle%5D)+AND+%22Proc.+SPIE%22%5BJournal%5D)">Kirigami design and fabrication for biomimetic robotics.</a> Proc. SPIE. 9055, Bioinspiration, Biomimetics, and Bioreplication. 90550G (2014).</li>
<li>Kim, T., Lee, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shape+transformable+bifurcated+stents%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Shape transformable bifurcated stents.</a> Scientific Reports. 8, 13911(2018).</li>
</ol>

The authors have nothing to disclose.

Stents are often used to clear the clogged internal pathways such as the blood vessels and airways of patients. Surgical operation of inserting stents requires the careful consideration of the patient&#8217;s illness and human anatomical characteristics. The shape of the vessel is complex, and diverse branching conditions exist. However, the standard stent operational procedures are based on mass-produced stents with standard sizes. In this protocol, we showed how to personally tailor the fabrication of the stent based on the exact geometry of the blood vessels. In doing so, we designed the stent so that the interior is made hollow and the surface tubular meshes will fold and return to the full unfolded configuration when activated. We have targeted bifurcated stents, which are typically used during operations with multiple numbers of tubular stents. The design of our bifurcated stents is performed as a whole, and a single operation is required regardless of how complex and how many branches exist in the branched vessels. The key enabling technique that we have used to solve the problem is the SMP. The ability of the structure to return to its original shape is anticipated, so forces are exerted to prevent the expanded pathways from the re-contraction.
Another important idea is the use of a kirigami structure. The most difficult part is how one can shrink the Y-shaped branches into a compact cylindrical tube. This problem has been solved using a kirigami structure. Each branch is folded into half cylinders and then merged together.
We found an optimal temperature of 220-230 &#176;C to memorize the bifurcated stent shape. Based on this fact, the extruder temperature was set to 230 &#176;C. When the temperature was set above this temperature, the accuracy of the shape was compromised. When the temperature is set below this temperature, the SMP clogged the 3D printer nozzle. If different materials are used, then the extruder temperature should be adjusted. The temperature of the printer bed was set at room temperature. We experienced unwanted deformation of the structure when the printer bed temperature was set higher. Additionally, it is recommended that the interior fill is set to above 70%. It is recommended to avoid or minimize the generation of supporters, as they will impose additional postprocessing burdens.
The glass transition temperature of the SMP used was 55 &#176;C, and the softening of the printed structure occurred above this temperature. When folding the printed bifurcated stent, we immersed the whole structure into a water heated bath above this temperature. When different SMPs are used, one should first find the glass temperature of the particular material. The recovery characteristics of other temperatures can be found in Kim and Lee15, where faster responses were shown for higher temperatures.
We used an FDM 3D printer to fabricate the bifurcated stent. The size of the produced stent was too large to be inserted into real human vessels. Researchers should consider using different types of 3D printers or 3D printers with smaller nozzle diameters. The latter is technically difficult because SMPs are often very viscous and will easily clog the nozzle, especially when smaller diameter-sized nozzles are used.
The limitations of our work are as follows. The glass transition temperature was too high to be used inside patients. Furthermore, this particular material was not proven to be biocompatible. It is also preferable that the stent be biodegradable when the vessel no longer needs the stent to support it from collapsing. These problems could be solved with the use of other types of SMPs and further extensive live experiments.

Stents are used to widen narrowed or stenosed passages in humans, such as blood vessels and airways. Stents are tubular structures that resemble the passages and mechanically support the passages from further collapsing. Typically, self-expanding metal stents (SEMS) are widely adopted. These stents are made from alloys composed of cobalt-chromium (stainless-steel) and nickel-titanium (nitinol)1,2. The downside of metal stents is that pressure necrosis can exist where the metal wires of the stent come into contact with the live tissues and the stents are impacted. Furthermore, the vessels of the body can be irregularly shaped and are much more complex than simple tubular structures. In particular, there are many specialized clinical procedures to install stents in branched lumens. In a Y-shaped lumen, two cylindrical stents are simultaneously inserted and joined at a branch3. For each additional branch, an additional surgical procedure needs to be conducted. The procedure requires specially trained doctors, and the insertion is extremely challenging due to the protruding features of the branched stents.
The complexity of the shape of bifurcated stents makes it a very suitable target for 3D printing. Conventional stents are mass produced in standardized sizes and shapes. Using the 3D printing fabrication methodology, it is possible to customize the shape of the stent for each patient. Because shapes are made by repeatedly adding layer-by-layer of the sectional shapes of the target object, in theory, this method can be used to fabricate parts of any shape and size. Conventional stents are mostly cylindrical in shape. However, human vessels have branches, and the diameters change along the tubes. Using the proposed approach, all these variations in shapes and sizes can be accommodated. Additionally, although not demonstrated, the used materials can also change within a single stent. For example, we can use stiffer materials where support is needed and softer materials where more flexibility is required.
The shape changing requirement of bifurcated stents calls for 4D printing, namely, 3D printing with the additional consideration of time. 3D printed structures formed using specialized materials can be programmed to change their shape by an external stimulation, such as heat. The transformation is self-sustained and requires no external power sources. One special material that is suitable for 4D printing is an SMP4,5,6,7,8,9, which exhibits shape memory effects when exposed to a material-specific triggering glass transition temperature. At this temperature, the segments become soft so that the structure returns to its original shape. After the structure is 3D printed, it is heated to a temperature slightly above the glass transition temperature. At this point, the structure becomes soft, and we are able to deform the shape by applying forces. While maintaining the applied forces, the structure is cooled down, becomes hardened and retains its deformed shape, even after the applied forces are removed. Subsequently, at the final stage, when the structure needs to return to its original shape, such as the moment when the structure reaches the target site, heat is supplied so that the structure reaches its glass transition temperature. Finally, the structure returns to its memorized original shape. Figure 1 illustrates the various stages previously explained. The SMPs can be easily stretched, and there are some SMPs that are biocompatible and biodegradable9,10. There are many uses for SMPs in the field of medicine9,10, and stents11,12 are one of them.
The patterns of the stents and the folding design follow the Japanese paper cutting design called &#34;kirigami.&#34;&#160;This process resembles the well-known paper folding technique called &#34;origami,&#34;&#160;but the difference is that in addition to folding, cutting of the paper is also allowed in the design. This technique has been used in arts and has also been applied in engineering applications2,3,13,14. In short, kirigami can be used to transform a planar structure to a three-dimensional structure by applying forces at specifically designed spots. In our design requirements, the stent needs to be a simple cylindrical shape when inserted into the pathways, and the cylinder should divide along its length where each half should unfold to a fully cylindrical shape at the targeted branched vessel. The solution lies in the fact that the main vessel and the side branches are folded into a single cylinder such that the side branches will not interfere with the walls of the vessels during the insertion. The unfolding command signal comes from the increase in the ambient temperature above the glass transition temperature of the SMP. Additionally, the folding will be conducted outside the patient body by softening the 3D printed bifurcated stent and folding the side branch into the main vessel.
Conventional methods required the insertion of multiple cylindrical stents whose number equals the number of branches. This method was inevitable because the protrusions of the side branches hampered the walls of the pathways and made it impossible to insert a complete bifurcated stent in its entirety. Using the kirigami structure and 4D printing, the above problems can be resolved. This protocol also shows the visualization of the effectiveness of the proposed method using a silicone vessel model fabricated after the shape of blood vessels. Through this mock-up, the effectiveness of the proposed invention during the insertion process and further possibilities of new applications can be seen.
The purpose of this protocol is to clearly outline the steps involved in printing an SMP using a fused deposition modeling (FDM) printer. Additionally, techniques involved in deforming the printed bifurcated stents to the folded state, the insertion of the folded bifurcated stents to the target site, and the signaling and unfolding of the structure to its original shape are given in detail. The demonstration of the insertion utilizes a silicone mock-up of blood vessels. The protocol also provides the procedures involved in fabricating this mock-up using a 3D printer and molding.

<table><tbody><tr><td>Fortus380mc</td><td>Stratasys</td><td>Fortus 380mc</td><td>FDM 3D printer for printing blood vessel mock-up</td></tr><tr><td>Moment1 3D printer</td><td>Moment</td><td>Moment 1</td><td>FDM 3D printer for printing bifurcated stent</td></tr><tr><td>PC(white) Filament Canister</td><td>Stratasys</td><td>PC(white) Filament Canister</td><td>PC filament for printing blood vessel mock-up</td></tr><tr><td>PLM software NX 10.0</td><td>Siemens</td><td>NX 10.0</td><td>3D CAD modeling software</td></tr><tr><td>Sandpaper</td><td>DAESUNG</td><td>CC-600CW</td><td>Smooting out the surface of the bifurcated stent&amp;nbsp;</td></tr><tr><td>Shape Memory Polymer filament</td><td>SMP Technologies Inc</td><td>MM-5520</td><td>Shape memory polymer filament</td></tr><tr><td>silicon</td><td>Shinetus</td><td>KE-1606</td><td>silicon for blood vessel mock-up</td></tr><tr><td>Simplify3D</td><td>Simplify3D</td><td>Simplify3D 4.0.1</td><td>Slicing software for model slicing&amp;nbsp;</td></tr></tbody></table>

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.

In this protocol, we showed the procedures required to fabricate a bifurcated stent. The stent uses a kirigami structure to allow the bifurcated stent to fold into a compact cylindrical tube, which is very suitable for sliding through the narrow pathways of blood vessels. The SMP allows the folded structure to return to its original shape when the temperature reaches the glass transition temperature. The original shape, 3D printed using the SMP material, closely matches the branched vessels. In other words, the interior surface of the branched vessels, where the bodily fluid is flowing, is offset further inside by the prescribed thickness of the fabricated stent. A solid form is created between the interior surface and the offset surface. This solid form exactly fits the vessel and can be used as a model for the stent. Due to the ability of the SMP to return to its memorized shape, the folded structure will return to the predeformed shape once heated above its glass transition temperature. The two branched stents can be easily formed into half-cylindrical tubes by taking advantage of the kirigami structure. The two halves of the cylinders are merged into one cylinder, and the united structure has been shown to slide through the main vessel and reach the bifurcation area. To return the folded structure to its original shape, the experiment was performed in a water at a temperature of 60 &#176;C. It has been shown that each side branch will divide, and each branch will go to its pairing vessels in the bifurcation area. The bifurcated stent was inserted into the Y-shaped vessels as a whole requiring only a single operation. This is much simpler than the conventional operation requiring insertions of each branching stent separately. These results show that it is possible to simplify the stent insertion operation to a single operation, whereas previous stent operations required the number of insertions of side branch stents to be the same as the number of side branching blood vessels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59746/59746fig1.jpg" /> 
Figure 1: Shape transformation diagram of the SMP. (A) The printed shape is the original shape. (B) When heated above the glass transition temperature (Tg), the structure becomes soft. When a force is applied, the structure is deformed to the desired shape. (C) The structure is fixed to a deformed shape by cooling. (D) When heated again above the glass transition temperature, a recovery force that returns the deformed shape to its original shape is generated. (E) The recovered shape is the same as the original shape. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig1large.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/59746/59746fig2.jpg" /> 
Figure 2: The names of the parts of a Y-shaped blood vessel are shown. Y-shaped vessels have a main vessel and a side branch. The main vessel consists of a proximal main vessel and a distal main vessel. The proximal main vessel is divided into the side vessel and the distal main vessel, which lies above the bifurcated core. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig2large.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/59746/59746fig3.jpg" /> 
Figure 3: Design of the blood vessel. (A) Right side view of the modeled blood vessel. This side is designed as a hook shape to express the three-dimensional nature of a real blood vessel in the human body. (B) Front view of the modeled blood vessel. Rotated view of the Y-shaped blood vessel according to Figure 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig3large.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/59746/59746fig4.jpg" /> 
Figure 4: Silicone blood vessel mock-up. A container made with acrylic plates and 3D printed blood vessel models are used as a mold to create this mock-up. The mock-up was made using liquid silicone, which was hardened after drying. The front view (A) and the side view (B) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig4large.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/59746/59746fig5.jpg" /> 
Figure 5: Design of the bifurcated stent&#8217;s branches using kirigami. (A) Conceptual design of the stent branch. The sheet is cut along the black line. Subsequently, external forces are applied at the specific points in the specified direction, as marked by the red arrows. The resulting geometry of the operations described in A is shown to the right, B. A planar sheet has been transformed into a three-dimensional tubular shape. (B) The design of a tubular stent based on the kirigami structure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig5large.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/59746/59746fig6.jpg" /> 
Figure 6: The three-dimensional model of the bifurcated stent. The trunk uses wavy patterns quite similar to the conventional stent design. The two upper branches utilize kirigami structures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59746/59746fig7.jpg" /> 
Figure 7: SMP filament. It is produced in a filament form that is easy to print using a commercial 3D printer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59746/59746fig8.jpg" /> 
Figure 8: Picture of a 3D printed bifurcated stent using an FDM (fused deposition modeling) 3D printer. The 3D printed bifurcated stent is attached to the 3D printer bed using a double-sided gluing tape to prevent the output from slipping. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59746/59746fig9.jpg" /> 
Figure 9: Postprocessing of the 3D printed result. (A) Removal of the supporters. The bifurcated stent is hollow in the interior and thus requires a supporter during 3D printing. The removal of the supporters is required. (B) The bifurcated stent with the supporters removed. (C) The bifurcated stent is spray-painted to clearly contrast it from the silicone pathways. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/59746/59746fig10.jpg" /> 
Figure 10: Illustration of the deformation and the recovery shape of the bifurcated stent. (A) The stent is heated to make it malleable. Subsequently, forces are applied to fold the branches into a half-cylindrical shape. (B) Half-cylindrical shapes are combined into a single tubular structure. The folding procedural steps are from the left to the right, and the recovery process is the reverse of the folding, which occurs from the right to the left. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/59746/59746fig11.jpg" /> 
Figure 11: The original and deformed state of the bifurcated stent. Notice the deformed shape is the shape of a cylinder and can be easily inserted into the trunk portion of the blood vessels. When the compactly folded shape is heated above the glass transition temperature, the shape returns to its original bifurcated shape. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/59746/59746fig12.jpg" /> 
Figure 12: The time lapsed shots of the recovering procedures of the folded stent inserted into the branched blood vessels are shown. (A) The procedural unfolding steps when the bifurcated stent is inserted into the Y-shaped vessels are shown. Initially, a single cylindrical tube is inserted. The inserted tube starts to divide once reaching the bifurcated core and returns to its unfolded original shape. (B) The timed images of the experiment. The upper left shows the insertion of the folded tube into the opening trunk of the vessel. The upper right shows the division of the inserted stent at the bifurcated core. The bottom row shows the recovery of the stent and the exact fit of the final bifurcated stent that perfectly fits the morphology of the targeting blood vessels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/59746fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/59746/59746bifurcated_stent.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/bifurcated_stent.stl" target="_blank">Please click here to download this file.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/59746/59746blood_vessel.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59746/blood_vessel.stl" target="_blank">Please click here to download this file.</a> 
Supplementary Files. Digital model of the vessel model.

Watch this Scientific Journal Video about 4D Printed Bifurcated Stents with Kirigami-Inspired Structures at JoVE.com

4D Printed Bifurcated Stents with Kirigami-Inspired Structures

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 ...

4d-printed-bifurcated-stents-with-kirigami-inspired-structures

Nanyang Technological University

Research

JoVE Journal

Engineering

7.9K Views.  Gwangju Institution of Science and Technology. Shape-memory polymeren kunnen worden geprogrammeerd om van vorm te veranderen wanneer aan bepaalde voorwaarden is voldaan. 3D-printers kunnen deze vormen produceren door lagen na lagen te stapelen, waardoor we elke denkbare vorm kunnen maken. In deze video laten we zien hoe we stents kunnen produceren die kunnen worden gekrompen tot een compacte vorm wanneer ze worden geleverd, en op het juiste moment kunnen herstellen naar de meest complexe vorm.Door het gebruik van deze methode is he...

Video: 4D Printed Bifurcated Stents with Kirigami-Inspired Structures

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Bioengineering

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Medicine

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

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 ...

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

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 ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds with adjustable properties. The aim of this study was to create multilayered scaffolds mimicking the architectural fiber characteristics of human heart valve leaflets using conductive 3D-printed collectors.
Models of aortic valve cusps were created using commercial computer-aided design (CAD) software. Conductive polylactic acid was used to fabricate 3D-printed leaflet templates. These cusp negatives were integrated into a specifically designed, rotating electrospinning mandrel. Three layers of polyurethane were spun onto the collector, mimicking the fiber orientation of human heart valves. Surface and fiber structure was assessed with a scanning electron microscope (SEM). The application of fluorescent dye additionally permitted the microscopic visualization of the multilayered fiber structure. Tensile testing was performed to assess the biomechanical properties of the scaffolds.
3D-printing of essential parts for the electrospinning rig was possible in a short time for a low budget. The aortic valve cusps created following this protocol were three-layered, with a fiber diameter of 4.1 &#177; 1.6 &#181;m. SEM imaging revealed an even distribution of fibers. Fluorescence microscopy revealed individual layers with differently aligned fibers, with each layer precisely reaching the desired fiber configuration. The produced scaffolds showed high tensile strength, especially along the direction of alignment. The printing files for the different collectors are available as Supplemental File 1, Supplemental File 2, Supplemental File 3, Supplemental File 4, and Supplemental File 5.
With a highly specialized setup and workflow protocol, it is possible to mimic tissues with complex fiber structures over multiple layers. Spinning directly on 3D-printed collectors creates considerable flexibility in manufacturing 3D shapes at low production costs.

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

Electrospinning has become a widely used technique in cardiovascular tissue engineering as it offers the possibility to create (micro-)fibrous scaffolds ...

4D Printed Bifurcated Stents with Kirigami-Inspired Structures

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