Our protocol allows researchers to generate hierarchical vessel networks within a fully engineered and implantable flap, which paved the way for new studies on blood vessel behavior under integration in vivo. This technique combined the versatility of two 3D printing technologies to create and assemble vessels on the micro and mesoscale that can be directly anastomosed with host vessels using microsurgery. Fully engineered vascularized flaps can be used to treat large tissue defects as well as to provide a platform for studying tissue developments and integration.
The proposed methods can be extended to study the incorporation of tissue-specific cells, as well as to evaluate the use of different bioink and support materials. Begin by filling in the BVOH mold with 30 microliters of the polymer solution. Centrifuge the mold at 100 times g for 2 minutes and top up the mold with a further 20 microliters of the polymer solution to ensure complete filling.
Freeze the filled molds at minus 80 degrees Celsius for at least 30 minutes to ensure all the polymer solution freezes. Remove the solvent from the molds by lyophilizing them overnight. To remove the sacrificial mold material, transfer the molds after the freeze-drying process to a 5-liter deionized water bath under gentle stirring.
Replace the water in the bath when it gets cloudy. When all the BVOH dissolves, air-dry the scaffolds and store them in a vacuum chamber until use. Transfer approximately 4 milliliters of the prepared, compacted support material into each well of a 12-well plate using a positive displacement pipette.
Tap the well plate on a hard surface to force the support material to spread evenly in the well. Prepare a suspension containing 2 million HAMEC-ZsGreen and 6 million dental pulp stem cells in 10 milliliters of endothelial cell medium. Centrifuge the cell suspension at 200 times g for 4 minutes to obtain a cell pellet, then aspirate the supernatant medium and resuspend the cell pellet in 1 milliliter of rhCollMA bioink to obtain a bioink with a total cell concentration of 8 million cells per 1 milliliter of bioink.
Fit a 0.22-millimeter internal diameter needle to a 3-milliliter amber printing cartridge and place the cartridge in a 50-milliliter conical tube. Transfer 1 milliliter of the cells-bioink mixture to the printing cartridge by filling it from the top using a positive displacement pipette to reduce bubbles. Install the printing cartridge in the appropriate tool at the bioprinter.
Sketch a 2D pattern of a 4-millimeter square containing a 2-millimeter diameter circular channel in its center. Extrude the sketch by 4 millimeters to obtain a 4-millimeter cube with a central channel. Export this object as a STL file.
Import a 12-well plate STL template into the solid modeling tab in the bioprinter slicing software and place it in the designated area of the virtual print bed. Import the STL file of the cube shape into the solid modeling tab in the bioprinter slicing software. Click on the use external slicer checkbox followed by configure slicer under the object property section.
In the pop-up window, choose a rectilinear pattern for the fill pattern and type 30%in the fill density, click accept. Click on the cube and move it using the mouse to place a copy of the cube shape in each desired virtual well. Click on the add material button in the materials tab of the bioprinter slicing software to create new material settings for rhCollMA bioink.
Choose the material settings for printing. For pressure, type 2 psi, and for speed type 20 millimeters per second in the corresponding boxes. Assign the line width and line height values to 0.24 millimeters, and the acceleration value to 400 millimeters per square second.
In the solid modeling tab, click on the cube object and then click on the rhCollMA material from the material section to assign it to the cube shape. In the bioassembly tab, click on the Send Print Job button to send the print job to the bioprinter. Load the printing cartridge loaded with the cellular bioink into the 3-milliliter ambient pneumatic dispense tool of the 3D extrusion-based bioprinter.
Click on the Cool button under the Control Heating or Cooling tab on the bioprinter interface screen to set the print bed temperature to 4 degrees Celsius. Load the plate with the support bath on the printer bed. In the Print tab on the bioprinter interface screen, click on the print job, then click on Start, followed by GO to begin the print job.
After printing, expose the well plate to a 405-nanometer light source with an intensity of 3 milliwatts per square centimeter for 30 seconds to initiate the crosslinking of the rhCollMA bioink. After crosslinking, incubate the well plate at 37 degrees Celsius and 5%carbon dioxide for at least 20 minutes until all the support bath melts. Gently aspirate the liquified support bath and replace it with the endothelial cell medium.
Incubate the constructs at 37 degrees Celsius and 5%carbon dioxide. Immediately after support material removal of the bioprinted microvascular network, place a fibronectin-coated PLLA:PLGA vascular scaffold next to the bioprinted structure. Insert the vascular scaffold in the main channel of the bioprinted structure.
Incubate the assembled constructs at 37 degrees Celsius and 5%carbon dioxide for two days. Prepare a cell suspension of tdTomato-expressing human adipose microvascular endothelial cells at a concentration of 10 million cells per milliliter. Place a 20-microliter droplet of this cell suspension on a hydrophobic surface.
Gently place the vascular scaffolds on top of the droplet such that the lumen of the scaffold on one end makes contact with the cell droplet. Aspirate the droplet from the opposing end of the scaffold filling its lumen with the cell suspension. Place the seated scaffold in a micro-centrifuge tube and put it in a rotator inside a humidified incubator for 60 minutes.
Finally, transfer the scaffold into a 12-well plate and add 2 milliliters of endothelial cell medium. The side view of a representative engineered flap imaged four days after the endothelial lining of the vascular scaffold is shown here. The bioprinted microvasculature is shown in green, while the endothelial lining is shown in red.
The representative image shows the anastomoses between the bioprinted vasculature and the endothelial lining. Immunostaining for the smooth muscle actin, nuclei, and endothelial cells after seven days of incubation is shown here. The representative image displays completed anastomoses of the engineered flap with a rat's femoral artery before the clamp removal and after the clamp removal.
This technique can be used to study the maturation and integration of 3D-printed vasculature in vivo and monitor the perfusion of the implant in the hind limb to establish the potential and safety for its use to treat tissue defects.
