This method can help answer key questions in the vascular tissue-engineering field such as monitoring the dynamics of engineer vascular growth and remodeling during long-term culture. The main advantage of using optical coherence tomography, or OCT, is that it is a readily available, realtime and nondestructive imaging strategy to characterize the structural features and remodeling process of engineered vessels. Demonstrating the procedure will also be Shangmin Liu, and Wentao Ma, technicians from my laboratory.
To begin this procedure, fabricate the PGA scaffold as described in the text protocol Dip PGA scaffolds in one mol-per-liter sodium hydroxide for one minute, to adjust the spacial structure of the mesh. Then, soak the scaffolds in tissue-culture-grade water three times for two minutes each. Each time, gently pat dry the scaffold with a tissue paper.
Then, dry up the scaffolds in a hood with a blower for one hour. To assemble the bioreactor and the y-junction for OCT imaging, first, soak the self-developed glass cylindrical bioreactor, PGA scaffolds, silicone tube, bio-compatible tubes, connectors, stir bar, and equipment for assembly in a 95 percent ethanol tank for two hours. Pull the PGA scaffold through the side arms of the bioreactor connected to one side with a connector, as well as to another side with the y-junction used to deliver the OCT guide wire.
Assemble another PGA scaffold in the bioreactor in the same way. Fit the polytetrafluoroethylene to the bioreactor lips by tightening it with 4-0 sutures. Put the bioreactor in the ethanol tank again for one hour before drying it overnight in a hood with the blower on.
Now, isolate human umbilical artery smooth muscle cells from human umbilical arteries by standard explant techniques In the cause of saving the cells into PGA scaffolds, avoid any dripping of cell suspension on the bottom of bioreactor. Also, cover the bioreactor with silicone-stopper lid as soon as possible after cell seating to prevent contamination. Expand and maintain the cells in smooth muscle growth medium.
Seed the human umbilical artery smooth muscle cells at a concentration of 5 million cells-per-milliliter in the above culture medium onto the PGA scaffolds. After placing a sir bar in the bioreactor, cover it the silicone stopper lid that has one feeding tube and three short tubing segments inserted for gas exchange. Attach PTFE 0.22 micron filters to each air change tube, and attach one heparin cap to the feeding tube.
Adjust the stir bar with a stirring speed of 13 rounds-per-minute. Then, assemble the glass bioreactor, silicone stopper lid, and PGA scaffold into the culture system. Allow the cells to adhere for 45 minutes by leaning the bioreactor every 15 minutes with a stand to the left and to the right.
Now, connect the LOO-OH-YEE pump, phosphate-buffered saline bag, and driver with the biocompatible tubes in order to create the profusion system. Open the driver to fill the tubes with PBS. Place the overall bioreactor in a humidified incubator with 5 percent CO2 at 37 degrees Celsius.
Fill the culture chamber with 450 milliliters of the culture medium. Grow the seeded scaffolds under static culture for one week, changing the culture medium every three to four days by aspirating half of the old medium through the feeding tube and refilling the reactor with an equivalent amount of fresh culture medium. To prepare the profusion system for OCT imaging, pump fluids in the PBS bag to circulate them through the biocompatible tubes and back to the bag.
Open the power of the driver and regulate the pump setting with a frequency of 60 beats-per-minute and an output systolic pressure of 120 millimeters of mercury. Adjust the mechanical parameters according to the needs of the tissue-engineering vascular culture. Click the run button to make the profusion system work.
Provide the fix pulsatile simulation to the vessels for three weeks by iteratively pressurizing the biocompatible tubes after one week of static culture. Use a light source to ensure the axial resolution of ten to twenty microns and the image depth of one to two millimeters to identify the structure of the tissue-engineered blood vessels, or TEVBs, based on the frequency domain OCT intravascular imaging system. Turn on the power switch and open the image capture software Connect the fiber-optic imaging catheter to the drive motor and optical controller, with the catheter automatic retreat function.
Set the parameters of the image acquisition rate to ten frames-per-second, with an automatic pullback speed of ten millimeters-per-second. Now, attach the imaging catheter to the y-junction via the heparin cap with an 18-gauge needle. Place the catheter into the silicone tube and identify the structure tightness of the PGA mesh before loading the PGA scaffold onto the bioreactor.
Now, place the catheter tip over the region of interest. Adjust the pullback device and check for the image quality. Acquire images at one, four, seven, ten, 14, 17, 21, and 28 days in culture for each individual TEBV.
Save these images sequentially with a realtime observation of the TEBV microstructure including surface morphology, internal structure, and composition. Repeat the measurement three times to get a reliable measurement of the engineered vessels each time. Capture a series of images throughout the testing using the image capture software.
To use image analysis software to measure the tissue-engineered blood vessel wall thickness, first select the image to be analyzed. Then, click the tracking tool to automatically identify the inner side of the tissue-engineered blood vessel with the software and manually sketch the outer side. A diagram of thickness will appear on the screen.
Repeat the measurement for five times to get a reliable measurement of the constructs. Consider using two independent investigators obligated to the obtain information. Open the silicone stopper lid placed over the bioreactor when the culture is finished, and discard the culture medium.
Loosen the polytetrafluoroethylene from the bioreactor lips and cut the silicone tubes from the outer side of the polytetrafluoroethylene with scissors. Harvest the TEBVs from the bioreactor, and cut them into sections for a scanning-electron microscopy examination. Pull out the supporting silicone tube, and fix the sections with 4 percent power formaldehyde.
Perform routine histological staining with masson's trichrome and sirious red to examine the morphology of collagen and PGA. To assess the PGA content and collagen component, observe histologic samples with sirious red staining through a polarized microscope. OCT images are compared with histopathological finds of TEBVs after four weeks of culture.
The OCT image shows compact microstructure and specific components compared with histological assessment. The culture medium, the silicone tube, the TEBV, and the PGA fragment are visible. Masson's trichrome staining demonstrates collagen fibers distributed in a certain direction, along with PGA remnants in the media layer of the engineered vessels.
Sirius red staining reveals PGA remnants and a collagen component by using a polarized microscope. Scanning-electron micrographs of engineered vessels demonstrate a compact microstructure. Meanwhile, this intraluminal imaging modality adopts nondestructive and easy monitoring of TEBVs, including the remodeling process and visual morphology in-situ in long-running culture.
As shown here, the remodeling occurs earlier and the morphological changes manifest more obviously in the dynamic group through comparison of TEBV thickness. This technique paves the way for researchers in the field of vascular tissue engineering to explore structure features and the long-term remodeling process of engineered vessels. Clear display of polymeric remnants by polarized microscopy combined with OCT imaging might be useful for assessing scaffold degradation.
