Name: fabrication of engineered vascular flaps using 3d printing technologies
Uploaded: 2022-05-19T15:00:00
Duration: 8 min 31 s
Description: Watch this Scientific Journal Video about Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies at JoVE.com

This project received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 Research and Innovation Programme (grant agreement no. 818808). rhCollMA was generously provided by CollPlant (Rehovot, Israel). The authors thank the Technion&#39;s Pre-clinical Research Authority for the assistance with animal care, as well as Janette Zavin, Galia Ben David, and Idan Redenski.

<ol>
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The authors have no conflicts of interest to disclose.

Engineering vascularized tissues is one of the main challenges of tissue engineering20. Current methods for creating engineered vascular tissue focus on creating self-assembled microvasculature21,22,23 or fabricating mesoscale vascular scaffolds24,25 and not on recreating a system of hierarchical vasculature, which can be perfused immediately and directly upon implantation26. In this work, we describe a protocol that makes use of two 3D printing modalities to fabricate a hierarchical vessel network composed of microscale and mesoscale vasculatures. The protocol combines a 3D bioprinted, self-assembled microvascular network with a mesoscale vascular scaffold, achieving an implantable, vascularized flap. Furthermore, this paper presents a protocol for directly anastomosing this flap to a rat&#39;s femoral artery.
3D bioprinting has gained interest in recent years due to its versatility over traditional tissue engineering techniques. While this protocol describes the generation of a microvascular network in rhCollMA bioink, the methods used can be applied with few modifications to many other bioinks from the plethora of studied and novel bioinks and support baths27,28. We chose to use rhCollMA as a bioink due to the abundance of type I collagen in the human ECM, providing a suitable environment for cell attachment. Moreover, it is produced recombinantly in plants and further modified with methacrylate groups, which allows for photopolymerization and the formation of stable 3D hydrogels29,30. Photocrosslinking was achieved by the addition of the photoinitiator LAP, which has been shown to be non-toxic and is activated by exposure to 405 nm blue light, reducing the possible phototoxicity of UV light. However, the use of photosensitive bioinks necessitates the use of a phenol red-free culture medium for the preparation of the bioink and the support material. Furthermore, the protocol describes the use of gelatin support material, which enables the high-fidelity extrusion of bioinks such as rhCollMA. Thus, it is critical to ensure the use of cold medium during its preparation and the cooling of the printer bed. Excessive heating might occur due to the light source used for crosslinking or from elevated ambient temperatures.
An extrusion-based bioprinter has been used here to create the bioprinted microvascular network, and there are currently many commercially available bioprinters that can generate similar constructs. Moreover, the proposed methods may be easily modified and applied to study different geometries, sizes, and infill patterns. In this work, a rectilinear infill pattern was chosen to create interconnected pores, and this can be printed relatively quickly with high fidelity.
Air bubbles introduce a significant challenge in extrusion bioprinting, especially inside support materials. Therefore, it is crucial to minimize the presence and formation of these bubbles by using positive displacement pipettes for the transfer of the support material, the preparation of the bioink-cell suspension, and their transfer to the printing cartridges.
In this work, human adipose-derived endothelial cells and dental pulp stem cells were used as supporting cells due to their relatively easy isolation from patients. Moreover, a total cell concentration of 8 x 106 cells/mL was chosen since this concentration has been shown to establish the most developed vascular networks16. While this protocol can be employed to generate microvasculature using different cell types and sources, as well as different bioinks, a calibration of cell concentration must be done to establish the best conditions for the development of the microvascular network. Furthermore, tissue-specific cells (i.e., myoblasts or osteoblasts) can be incorporated within the bioink to achieve tissue-specific vascularized flaps.
The mold for the porous vascular scaffold was fabricated using 3D printed water-soluble material on a commercially available extrusion 3D printer. This results in a cost-effective technique based on rapid prototyping platforms, such that many different geometries and sizes of vascular scaffolds can be studied and screened rapidly31. Nevertheless, a limitation of this method is the resolution limit of most 3D printers32. However, with the rapidly evolving industry surrounding additive manufacturing, these limits are expected to improve over time. The use of organic solvents for the fabrication process is another limitation of the protocol, as most organic solvents are toxic to cells, preventing the ability to combine the bioprinting procedure with the vascular scaffold fabrication process.
The described method of seeding the lumen of the scaffold using aspiration as opposed to pushing the cell suspension has major effects on the localization of the seeded cells. Using negative pressure allows for endothelization of the inner lumen of the scaffold while minimizing any spilling of the cell suspension through the perforations on the scaffold&#39;s wall16.
The described &#34;cuff&#34; method for microsurgical anastomoses can be easily modified and adapted to different vascular scaffold materials or sizes, as well as to different arteries and veins in a wide scale of animal models. The adaptations to the protocol would include different polyimide tube sizes and suture sizes. This method does not require the perforation of the scaffold wall, which might lead to the development of defects. This work presents a protocol that can be expanded to many applications. The critical aspects of this protocol, which include the fabrication of meso- and microscale vasculature and their assembly and implantation, represent critical aspects of engineered flaps both for reconstructive applications, as well as vascular and other tissue engineering studies.

Severe tissue defects are caused by traumatic injuries, congenital defects, or disease, and the current gold standard for treating these defects is by using autologous grafts, vascularized tissue flaps, and microvascular free flaps as tissue substitutes. However, these options have the drawbacks of limited donor site tissue and donor site morbidity1. Thus, there is a growing demand for alternative tissue substitutes that can be used to correct these defects2. The thickness of the engineered tissue constructs is limited by the diffusion of nutrients and gases toward the cells, and, therefore, a proper vascular network is essential for generating large, thick, and properly nourished scaffolds.
Several approaches have been applied to promote the vascularization of engineered implants3, including the in vivo recruitment of vascular support from the host, the delivery of growth factors and cytokines within the scaffolds, the prevascularization of implants, the generation of a perfusable branching microvessel bed using micropatterning techniques4, the use of sacrificial materials for vascular channel/network formation5, as well as the creation of channels within 3D bioprinted constructs5,6. Vascularization of thick tissues requires the incorporation of a hierarchical vascular network consisting of macro-scale and microcapillary-scale vessels. The macro-scale vessels distribute blood effectively throughout the construct and allow for microsurgical anastomoses with the host blood vessels, while the microcapillary-scale vessels allow for nutrient diffusion.
Bioprinting has gained strong attention in recent years due to the advantages it offers over conventional tissue engineering methods. Tissues and organs are complex and intricate 3D objects with a specific architecture. 3D bioprinting, with its ability to deposit layers of biomaterials in high resolution, enables the capability of creating complex tissue and organ substitutes (e.g., kidney, lung, liver)7. Several printing technologies have been adapted for bioprinting, including extrusion-based, inkjet8, laser-assisted deposition9,10, and stereolithography-based11,12 bioprinting. The extrusion-based technologies rely on extruding the material through a nozzle by applying pressure on the material bulk surface opposite to the nozzle.
Free-form reversible embedding of suspended hydrogels (FRESH) is a bioprinting technique13,14 that uses a granular support material in which the extruded material is deposited and fixed in place by the support bath. The support bath gives mechanical support for the extruded, pre-crosslinked bioink until its crosslinking. The main advantage of this technique is that the support bath allows for extruding low-viscosity materials that cannot maintain their shape after extrusion and before crosslinking15. This expands the pool of available materials that can be used as bioinks.
This paper presents a protocol for the generation of a vascularized flap that combines microscale and mesoscale vasculatures. To achieve this, bioprinted, self-assembling, microvascular networks are generated in recombinant human collagen methacrylate (rhCollMA) hydrogel, which then connects to the interior of a larger, implantable, vascular scaffold, resulting in a fully engineered tissue flap16. To establish rapid and direct perfusion of engineered tissues, a direct microsurgical anastomosis to host vessels is required. The vascular scaffold does not have sufficient suture retention strength to be anastomosed using traditional microsurgical vessel wall suturing. Therefore, we describe a &#34;cuff&#34;17,18,19 method to achieve an anastomosis with a rat&#39;s common femoral artery. In this method, the vessel ends are secured with circumferential sutures, without the need to perforate the vessel wall.
Although the proposed protocol has been prepared to study hierarchical vasculature in the rhCollMA environment, this approach can be expanded and applied to a variety of new applications. The protocol can be applied to bioprinting various tissue-specific cells in different bioinks. Moreover, the geometry and size of the constructs can be easily modified to fit specific requirements, such as large tissue reconstruction or biological studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-Dioxane</td>
			<td>Biosolve Chemical</td>
			<td>42405</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 0.5&#34; blunt tip dispensing needles</td>
			<td>CML supply</td>
			<td>901-27-050</td>
			<td></td>
		</tr>
		<tr>
			<td>3cc amber syringe barrel &#38; piston set</td>
			<td>Nordson EFD</td>
			<td>7012085</td>
			<td>Amber syringes used to block light and prevent premature crosslinking</td>
		</tr>
		<tr>
			<td>5-0 AssuCryl PGA absorbale suture</td>
			<td>Assut Sutures</td>
			<td></td>
			<td>Absorbable sutures used for skin wound closure</td>
		</tr>
		<tr>
			<td>6-0 polypropelene sutures</td>
			<td>Assut Sutures</td>
			<td>9351</td>
			<td></td>
		</tr>
		<tr>
			<td>Acland clamps</td>
			<td>S&#38;T</td>
			<td>B-1V</td>
			<td></td>
		</tr>
		<tr>
			<td>Adventitia scissors</td>
			<td>S&#38;T</td>
			<td>SAS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled no.3 jeweler&#39;s forceps</td>
			<td>S&#38;T</td>
			<td>JFAL-3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>BioAssemblyBot 400 3D Bioprinter</td>
			<td>Advanced Solutions</td>
			<td></td>
			<td>a 6-axis 3D bioprinter</td>
		</tr>
		<tr>
			<td>Bovine albumin serum Probumin</td>
			<td>Millipore</td>
			<td>&#160;82-045-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>vetmarket</td>
			<td>B15100</td>
			<td></td>
		</tr>
		<tr>
			<td>BVOH filament</td>
			<td>Verbatim</td>
			<td>55903</td>
			<td>a water-soluble 1.75 mm diameter filament</td>
		</tr>
		<tr>
			<td>Clamp applying forceps</td>
			<td>S&#38;T</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental pulp stem cells</td>
			<td>Lonza</td>
			<td>PT-5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dietrich bulldog clamps</td>
			<td>Fine Science Tools (FST)</td>
			<td>18039-45</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate (Na2HPO4)</td>
			<td>Carlo Erba Reagents</td>
			<td>480141</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>S&#38;T</td>
			<td>18039-45</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, No Phenol Red</td>
			<td>Sartorius</td>
			<td>01-053-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Duratears</td>
			<td>Alcon</td>
			<td>DJ03</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM media + bullet kit</td>
			<td>Sciencell</td>
			<td>#1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Gadot-Group</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>glutamine substitute</td>
		</tr>
		<tr>
			<td>Goat anti-mouse Cy3 antibody</td>
			<td>Jackson</td>
			<td>115-166-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium&#160; 5,000 I.U./mL</td>
			<td>Panpharma</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Human adipose microvascular cells</td>
			<td>Sciencell</td>
			<td>#7200</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Sigma</td>
			<td>F0895-5MG</td>
			<td>A stock concentration of 1 mg/mL</td>
		</tr>
		<tr>
			<td>Isoflurane, USP Terrell</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-011-25</td>
			<td></td>
		</tr>
		<tr>
			<td>LifeSupport</td>
			<td>Advanced Biomatrix</td>
			<td>5244</td>
			<td>a gelatin support slurry for FRESH 3D bioprinting</td>
		</tr>
		<tr>
			<td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
			<td>Sigma-Aldrich</td>
			<td>900889</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-glucose DMEM</td>
			<td>Biological Industries</td>
			<td>01-050-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>MICROMAN E M1000E, 100-1,000 &#181;L</td>
			<td>Gilson</td>
			<td>FD10006</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SMA antibody</td>
			<td>Dako</td>
			<td>M0851</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco</td>
			<td>11140068</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>ChemCruz</td>
			<td>&#160;SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Nystatin Solution</td>
			<td>Biological Industries</td>
			<td>03-032-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene oxide), M.W. 250,000 to 400,000</td>
			<td>Acros Organics</td>
			<td>178602500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(L-lactic acid), IV 5.0 dl/g (PLLA)</td>
			<td>Polysciencse, Inc.</td>
			<td>18582-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide tubing, ID: 0.0249&#34;, OD: 0.0273&#34;</td>
			<td>Cole-Parmer</td>
			<td>95820-05</td>
			<td>A thin-walled tube used to fabricate cuffs for microsurgical anastomoses</td>
		</tr>
		<tr>
			<td>Prusa I3 MK2.5 3D Printer</td>
			<td>Prusa Research</td>
			<td>http://www.prusa3d.com/</td>
			<td>a popular commercial 3D printer</td>
		</tr>
		<tr>
			<td>Resomer&#160;RG 503 H, Poly(D,L-lactide-co-glycolide) (PLGA)</td>
			<td>Evonik Industries</td>
			<td>719870</td>
			<td></td>
		</tr>
		<tr>
			<td>rhCollMA</td>
			<td>CollPlant</td>
			<td>https://collplant.com/</td>
			<td>generously provided by CollPlant (Rehovot, Israel)</td>
		</tr>
		<tr>
			<td>round-handled needle holder</td>
			<td>S&#38;T</td>
			<td>B-15-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle - #3</td>
			<td>Fine Science Tools (FST)</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>small fine straight scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Biosolve Chemical</td>
			<td>19030591</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic (NaH2PO4)</td>
			<td>Riedel-de Haen</td>
			<td>4276</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks</td>
			<td>Dassault Systems</td>
			<td></td>
			<td>CAD software</td>
		</tr>
		<tr>
			<td>Straight no.3 jeweler&#39;s forceps</td>
			<td>S&#38;T</td>
			<td>JF-3-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight serrated forceps</td>
			<td>Fine Science Tools (FST)</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scalpel Blade No.15</td>
			<td>Swann-Morton Limited</td>
			<td>305</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>BioLab LTD</td>
			<td>57836</td>
			<td></td>
		</tr>
		<tr>
			<td>TSIM</td>
			<td>Advanced Solutions</td>
			<td></td>
			<td>3D slicing and design software for the BioAssembly Bot</td>
		</tr>
		<tr>
			<td>Vessel dilator</td>
			<td>S&#38;T</td>
			<td>D-5a.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Tivato 700 surgical microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This protocol describes the fabrication of an engineered flap composed of a vascular scaffold (Figure 1Ai) and a bioprinted microvasculature (Figure 1Aii), which were assembled to achieve mesoscale and microscale vasculatures (Figure 1Aiii). Following the protocol, BVOH molds of the vascular scaffold were designed and 3D printed (Figure 1B,C). The obtained printed structures were visually inspected for small strands of BVOH, which might be found in the empty spaces of the molds (Figure 1D). These strands usually indicate incorrect material settings or that the BVOH has absorbed moisture. These strands should be removed as they might lead to incomplete filling of the mold and structural defects in the resulting vascular scaffold. Next, the molds were filled with PLLA:PLGA solution, followed by the freeze-drying process and the washing steps, as described in the protocol. The obtained PLLA:PLGA vascular scaffold was visually inspected to verify vessel wall integrity and lumen patency (Figure 1E).
A neutralized rhCollMA bioink at a concentration of 10 mg/mL was prepared and combined in a 1:1 ratio with the PEO:LAP solution. Human adipose microvascular endothelial cells labeled with Zs-Green1 and dental pulp stem cells were resuspended with the rhCollMA bioink, and the solution was loaded into a printing cartridge and onto the printer. Box shapes with a central channel with a rectilinear pattern (Figure 1D) were bioprinted inside the gelatin support bath. Following printing, the constructs were crosslinked, and the support bath was dissolved and washed. After 4 days of culture, the constructs were live-imaged to check for microvascular network self-assembly. Figure 1D shows an example of a highly developed HAMEC-ZsGreen1 microvascular network in the bioprinted construct.
Next, a fibronectin-coated vascular scaffold was inserted into the central channel of the printed construct (Figure 2A). The assembled constructs were cultured for 2 days, during which the cells contract the gel, attaching it firmly to the vascular scaffold. Then, the vascular scaffold was lined with HAMECs expressing tdTomato, according to the protocol. After 7 days of culture, the constructs were fixed and imaged. Figure 2B shows a side view of the assembled constructs where the endothelial cells in the bioprinted microvasculature are depicted in green, while the endothelial lining of the vascular scaffold is depicted in red. The image shows a green microvascular self-assembly in the bioprinted gel, while the vascular scaffold is lined with red endothelial cells. With higher magnification, sprouts originating from the red endothelial lining are seen sprouting and anastomosing with the bioprinted microvascular network (Figure 2C). Next, the constructs were stained for &#945;-smooth muscle actin (SMA) as a marker for the dental pulp stem cells. Following the immunostaining, the constructs were imaged using a laser-scanning confocal microscope (Figure 2D).
Lastly, after 7 days of culture, the engineered flaps were microsurgically anastomosed to a rat&#39;s femoral artery, as described in the protocol. A video of a representative procedure can be seen in Supplemental Video S1. Figure 2E shows a representative image of a completed anastomoses prior to clamp removal, and Figure 2F shows a representative image of the anastomoses site after clamp removal and hemostasis. There should be no bleeding visible prior to wound closure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63920/63920fig01.jpg" /> 
Figure 1: Representative images of the fabricated meso- and microscale vessels. (A) Schematic overview of the protocol&#39;s steps. Reproduced with permission16. (B) CAD design for the sacrificial mold of the vascular scaffold. (C) Side view of a representative 3D printed sacrificial mold (scale bar = 0.5 mm). (D) Top view of sacrificial mold (scale bar = 0.5 mm) (E) Representative vascular scaffold fabricated using the describe protocol (scale bar = 5 mm). (F) CAD design for the 3D bioprinted rhCollMA microvascular network. Grid lines = 1 mm. (G) Representative image of a highly developed bioprinted vascular network showing HAMEC-ZsGreen1 in green. Scale bar = 200 &#181;m. Abbreviations: CAD = computer-aided design; rhCollMA = recombinant human collagen methacrylate; HAMEC = human adipose microvascular endothelial cells; PLLA = poly-L-lactic acid; PLGA = polylactic-co-glycolic acid. <a href="https://www.jove.com/files/ftp_upload/63920/63920fig01large.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/63920/63920fig02v2.jpg" /> 
Figure 2: Representative images of the assembled vascularized flap. (A) A photograph of a representative assembly of bioprinted microvasculature and the vascular scaffold. Scale bar = 1 mm. (B) Side view of a representative engineered flap imaged 4 days after endothelial lining of the vascular scaffold. The bioprinted microvasculature is shown in green (HAMEC-ZsGreen1), while the endothelial lining is shown in red (HAMEC-tdTomato). Scale bar = 1 mm. (C) Representative image of anastomoses between the bioprinted vasculature in green and the endothelial lining in red. Scale bar = 200 &#181;m. (D) Representative image of immunostaining for smooth muscle actin (red), nuclei (blue), and endothelial cells (green) after 7 days of incubation. Scale bar = 0.1 mm. (E) Representative image of a completed anastomoses of the engineered flap with a rat&#39;s femoral artery before clamp removal and (F) after clamp removal. Abbreviations: HAMEC = human adipose microvascular endothelial cells; SMA = smooth muscle actin; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www.jove.com/files/ftp_upload/63920/63920fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video S1: Representative microsurgical procedure for anastomosing the vascular scaffold to a rat&#39;s femoral artery. <a href="https://www.jove.com/files/ftp_upload/63920/supplementary video 1 (1).mp4" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies at JoVE.com

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

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 ...

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JoVE Journal

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