This video shows how to fabricate porous tubular scaffolds and subject them to dynamic mechanical conditioning for arterial tissue engineering. This is accomplished by first fabricating the scaffolds using a biodegradable elastomer and assault fusion method. The scaffolds are then prepared for cell seeding and assembled into a bioreactor system.
In the bioreactor vascular smooth muscle cells seed the scaffolds and are cultured. Weeks later, tissue is harvested and analyzed using scanning electron microscopy, h and e staining and elastin autofluorescence. The resulting smooth muscle is both multilayered and perpendicularly oriented.
The main advantage of this technique existing like particulate bleaching, is to use the glass mold to fabricate it porous tubular scaffold and hyaluronic acid. As a mold deletes, The scaffolding mold is prepared from a piece of glass tubing. Put the tubing in a holder and pour hyaluronic acid solution prepared the day before into the tube.
The hyaluronic acid flow slowly down along the inner wall. When it reaches the bottom of the mold, flip the mold over. Repeat this step until the inner wall of the mold is evenly coated by the solution.
After coating, dry all the prepared glass molds in a vacuum oven at 37 degrees Celsius for 24 hours. Typically, four molds are prepared concurrently while the glass molds dry. Prepare the salt particle pours, grind and SVE salt particles to 25 to 32 micrometers.
The next day, assemble the prepared glass mold mandrel, PTFE tubing, heat shrinkable sleeve and PTFE ring. First, load the mandrel into 65 millimeter lengths of PTFE tubing and bake them at 120 degrees Celsius for five minutes. To shrink the PTFE while waiting pre-war a hybridization incubator at 37 degrees Celsius for at least 30 minutes.
Once the tubing has encased the mandrels, push the heat shrink sleeve onto the mandrel so it moves freely. Then place the mandrel inside the glass mold and attach A-P-T-F-E ring to the bottom of the mandrel. Check that the PTFE ring is snugly fit to the bottom of the glass mold.
Next, using a spatula and silicon rubber funnel, add salt particle porridges to the glass mold. Then tap the mold gently with the spatula for even particle distribution and scrape off the excess salt. Now turn off the warmed incubator and quickly load it With the salt packed molds, the salt will fuse over the next 30 minutes, after which dry the molds in a vacuum oven at 37 degrees Celsius for 24 hours.
The next day after cooling, remove the stainless steel mandrel from the molds by pushing it out while securing the PTFE ring. If needed, use needle nose pliers. Then remove the PTFE ring from the bottom of the mold.
Next, bake the molds to shrink the sleeve and remove the shrunken sleeves from the molds. Allow the molds to cool until they are used and store them in a desiccate in a hood. Using apo dropper angled the glass molds at 45 degrees and drop PGS solution into their inner lumen while slowly rotating the mold.
Check if the PGS solution flows down along the wall of the mold. If there is a dry spot, add more PGS Now allow the THF to evaporate in the hood for at least 30 minutes. Once the THF is gone, cure the molds in a vacuum oven.
After curing for one day, cool the molds to room temperature and slowly, vertically dip them into deionized water at 24 degrees Celsius. Tipping them too quickly creates air bubbles, which tear the scaffold. Carefully transfer the molds to the water bath.
Position them at an angle using a silicone tube, and allow the hyaluronic acid to dissolve over an hour. If after an hour the hyaluronic acid has not released from the mold, then with the mold still submerged, use a spatula to slowly push the hyaluronic acid off from both ends, and then slowly shake the mold. Now, check that the scaffolds haven't moved inside the glass mold, and if so, pull the scaffold slowly with forceps to release it from the mold.
Next, leach out the salt particles by carefully transferring the delicate scaffolds to a deionized water bath with gentle stirring. This will take at least three days and requires the water to be changed daily After the salt has all been leached out, transfer each scaffold to a 15 milliliter centrifuge tube filled with deionized water and freeze them in a dry ice box for an hour. Place the frozen centrifuge tubes into a lyophilizer for three days with the caps open.
Once freeze dried, store the scaffolds in a desiccate until they are used. Begin by cutting the scaffolds in 25 to 30 millimeter lengths. Next, prepare two silicone rubber stoppers by feeding PTFE tubing through the middle holes of each stopper.
Then cut one and a half millimeter lengths of hs ring and slide a length onto one end of the scaffold. Push one PTFE tube attached to the stopper into the same end of the scaffold with just enough overlap to be under the HS ring to firmly connect the scaffold to the tubing, shrink the HS ring in an oven and allow the assembly to cool down to room temperature. Now, a 50 millimeter polycarbonate tube, which functions as the bioreactor chamber is slipped over the scaffold and secured at the inner surface of the silicone rubber stopper.
Just as before another PTFE tube and stopper are secured at the other end of the scaffold with an hs ring to complete the chamber. The second stopper is attached to the other end of the polycarbonate tube. Next, the outer surfaces of the stoppers are attached to two aluminum alloy plates.
Feed two threaded rods into the side hole on each plate and secure the plates with thumb screws. Attach scaffold to the bioreactor. Now measure the seeable length of each scaffold, which is the distance between two hs rings, and calculate their inner surface areas.
For cell seeding. In an autoclave, sterilize the chambers each wrapped individually in foil along with each part of the bioreactor unit. Once sterilized, assembled the bioreactor inside of a cell culture hood.
Pre-treat and rinse the scaffold with a series of perfusions by using a peristaltic pump at one millimeter per minute in the flow circuit. Begin by rinsing with 70%ethanol, 50%ethanol, and then 25%ethanol for an hour each. Follow the three ethanol rinses with a two hour PBS rinse.
Finally, perfuse the bioreactor with SMC culture medium for 24 hours after which it is ready for cell seeding and experimentation. Now following the instructions in the accompanying manuscript, seed the bioreactor with 2 million cells per centimeter squared and over the next 21 days, change tubing media and make adjustments to the pump speed. Gradually, the pressure in the construct will increase from about four millimeters mercury on the first day of culture to over 100 millimeters mercury after two weeks in culture, after three weeks in culture, harvest the cells and prepare them for analysis as specified in the accompanying manuscript.
These tubular PGS scaffolds were fabricated by the salt fusion method. Scanning electron micrographs demonstrated that all of the scaffolds had homogeneous wall thicknesses and there were no partial defects at their cross-sections. Randomly distributed macro and micropores were observed on the luminal surface of all scaffolds.
After cell culture, multilayered SMCs grew with a perpendicular orientation to flow direction. Furthermore, the cells and DCM proteins completely covered the lumen of all PGS constructs. Elastin autofluorescence also showed circumferentially organized elastic fibers at the luminal surface of the construct.
After watching this video, you should have understand of how to make porous tubular scaffold, see the cells and culture scaffold using pre-designed bioreactor.
