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><tbody><tr><td>1,4-Dioxane</td><td>Biosolve Chemical</td><td>42405</td><td></td></tr><tr><td>27 G x 0.5&quot; blunt tip dispensing needles</td><td>CML supply</td><td>901-27-050</td><td></td></tr><tr><td>3cc amber syringe barrel &amp;amp; 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&amp;amp;T</td><td>B-1V</td><td></td></tr><tr><td>Adventitia scissors</td><td>S&amp;amp;T</td><td>SAS-15</td><td></td></tr><tr><td>Angled no.3 jeweler&#039;s forceps</td><td>S&amp;amp;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>&amp;nbsp;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&amp;amp;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 (Na&lt;sub&gt;2&lt;/sub&gt;HPO4)</td><td>Carlo Erba Reagents</td><td>480141</td><td></td></tr><tr><td>Dissection scissors</td><td>S&amp;amp;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&amp;nbsp; 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 &amp;micro;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>&amp;nbsp;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&quot;, OD: 0.0273&quot;</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&amp;nbsp;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&amp;amp;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 (NaH&lt;sub&gt;2&lt;/sub&gt;PO4)</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&#039;s forceps</td><td>S&amp;amp;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&amp;amp;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 ...

fabrication-engineered-vascular-flaps-using-3d-printing

Research

JoVE Journal

Bioengineering

3.7K Views.  Technion-Israel Institute of Technology. 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.

Video: Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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 ...

Novel Process for 3D Printing Decellularized Matrices

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

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

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

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

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

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

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

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

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 ...

Medicine

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...