Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Clinical applications of tissue-engineered trachea have been limited due to graft stenosis and delayed epithelialization. A mouse model of orthotopic tracheal replacement permits the study of the cellular mechanisms driving regeneration. Long-segment defects of the airway can exceed what current therapies are able to cure.

The tissue-engineered trachea offers the potential to create a replacement organ for these conditions. This model can be applied to animals with transgenic and lineage tracing properties. We can then modulate the host factors contributing to graft regeneration.

One of the key challenges in producing the scaffolds will be measuring and determining that the electrospinning parameters are the same each time. So every time you produce a scaffold, you need to measure the tip-to-collector distance, the humidity, and the temperature to ensure the scaffolds are the same each time. Critical to the success of this model is refinement of the microsurgical techniques and maintenance of the correct plane of anesthesia to permit spontaneous ventilation throughout the procedure.

Since the host trachea has an internal diameter of around one millimeter, graft and native tissue handling and suture placement are important and best demonstrated visually. Begin by dissolving eight weight percent polyethylene terephthalate, or PET, in hexafluoroisopropanol and heating the resulting solution to 60 degrees Celsius. While the solution is being warmed, dissolve three weight percent polyurethane, or PU, in hexafluoroisopropanol, combining the solutions once the PET solution has cooled.

Load a 60-milliliter syringe equipped with a 20-gauge blunt-tip needle with the polymer mixture. Use the syringe and a high-voltage DC power supply set to positive 14 kilovolts on the needle, another high-voltage DC power supply set to minus three kilovolts, a five-milliliter-per-hour flow rate, and a 20-centimeter tip-to-substrate distance to electrospin the polymer onto a one-millimeter diameter stainless steel rod being turned at 350 rotations per minute. Continue electrospinning until a 300-micrometer scaffold wall thickness is achieved.

Then, slide the scaffold off the rod, place the scaffold under vacuum overnight to remove any residual solvent, and sterilize the scaffold with an ultraviolet light dosage of 350 millijoules per centimeter squared for about 15 minutes. Under sterile conditions, remove the skin from the hind limbs of a six to eight-week-old, female, C57-Black/6 mouse with fine scissors and micro-Adson forceps, and use Dumont number five forceps and Dumont number 5/45 forceps to remove the fascia and the tendons. Separate the bones to allow each of them to be cut on both ends, and use a five-milliliter syringe equipped with a 25-gauge needle to flush the bone marrow into a Petri dish containing 30 milliliters of RPMI medium.

When all of the bones have been flushed, filter the bone marrow through a 70-micrometer nylon cell strainer into a 50-milliliter conical tube, and transfer the tube to a biosafety cabinet. Gently add the filtered mononuclear cell solution down the side of a 15-milliliter tube containing five milliliters of polysucrose and sodium diatrizoate without mixing the layers, and separate the plasma from the white and red blood cells by density gradient separation. At the end of the centrifugation, collect the middle clear layer consisting of the bone marrow mononuclear cells, and wash the bone marrow mononuclear cells at a one-to-one ratio in PBS by centrifugation.

Resuspend the pellet in five milliliters of PBS for a second wash, followed by resuspension in 10 milliliters of fresh RPMI medium for counting. Then, collect the cells with an additional centrifugation, and dilute the cells to a one times 10 to the seven cells per five microliters of fresh RPMI concentration. Before seeding the scaffold, cut the polymer to a five-millimeter length as necessary, and wet the scaffold with five microliters of RPMI medium for five minutes.

At the end of the incubation, remove the excess medium, and load the scaffold lumen with five microliters of the bone marrow mononuclear cell solution for 10 minutes. Then, insert a 21-gauge needle through the lumen of the scaffold, and place the graft in one milliliter of RPMI medium overnight in a 37-degree Celsius incubator. After induction of general anesthesia, confirm a lack of response to toe pinch in the recipient animal, and apply ointment to the animal's eyes.

Clip the hair on the surgical site from chin to clavicles, and place the animal on a surgical pad in a dorsal recumbent position. Using aseptic technique, disinfect the surgical site with a sequential povidone-iodine, 70%ethanol, povidone-iodine wipe series, and move the mouse under a dissecting microscope with the head at 12 o'clock. Use fine scissors and micro-Adson forceps to make a midline incision from the clavicles to the hyoid bone, and use Dumont number five and number seven fine forceps and a sterile cotton swab to clean the fascia before inserting a self-retaining Colibri retractor into the incision.

Use the forceps to open the strap muscles to expose the thyroid cartilage, cricoid cartilage, and trachea, and bluntly separate the trachea from the recurrent laryngeal nerves running parallel on either side of the tissue, followed by circumferential separation of the trachea from the esophagus. Using a 20-gauge needle and surgical marker, stain the anterior portion of the trachea. Make an incision below the third tracheal cartilage ring using a pair of Vannas-Tubingen spring scissors and a pair of Dumont number seven fine forceps to resect a three-ring section of trachea.

Holding the trachea with the fine curved forceps, use a sterile 9-0 nylon suture to secure the distal end of the trachea to the sternum to create a temporary tracheostomy, and use the 20-gauge needle and surgical marker to stain the anterior portion of the graft. To implant the graft, use a 9-0 sterile nylon suture, Dumont number seven fine forceps, and a needle holder to insert the proximal-posterior, proximal-lateral, and proximal-anterior stitches, and turn the animal 180 degrees. When the mouse is in position, release the temporary tracheostomy, and make a lateral incision five millimeters away from the anterior portion of the graft to facilitate the distal anastomosis.

To complete the distal anastomosis, place the sutures in a similar fashion to the proximal anastomosis, and re-approximate the graft position and strap muscles. Then, close the incision with 9-0 sterile nylon sutures in a running pattern, and place the animal alone in a recovery cage placed on a heating pad with monitoring until full recovery. Two weeks after implantation, a few basal epithelial cells can be observed by basal cell keratin five and 14 staining in tissue-engineered tracheal graft tissue sections.

Basal cells are also detected on the luminal surface of the graft at seven days after implantation. Upon explantation and histological analysis, graft stenosis has been identified as the main contributing factor in graft-implanted mice demonstrating signs of respiratory distress and stridor. Note that telescoping of the graft and native trachea is a common finding as a result of the surgical technique.

Staining for F4/80 reveals host macrophages within the stenotic region of the graft in conjunction with the presence of the basal epithelial cells. Avoid manipulation of the recurrent laryngeal nerves, and ensure that the proximal and distal sutures are in place to allow an airtight seal at the anastomosis. As this protocol deals with bone marrow mononuclear cells derived from biosafety level one agent mice, it is important to wear appropriate personal protective equipment to avoid contamination.