Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

Circumferential esophageal repair is technically challenging in a rat model, necessitating the development of tissue-engineered esophagus that enables the regeneration of esophageal mucosa and muscle without esophageal anastomosis leakage. We have implemented a two-layered tubular scaffold as a pilot model, consisting of inner nanofiber and an outer 3D-printing strand in conjunction with a microvascular anastomosis to minimize saliva leakage after transplantation. Conventional standard esophageal reconstruction is associated with wide range of complications and morbidity.

Therefore, esophageal tissue engineering may be a promising alternative strategies for developing a native patient esophagus models. The combination of hybrid scaffold, bioreactor cultivation, and an oral nutrition system can be applied to any system that has a similar contaminated environment with its continuous mechanical pressure. Begin by sterilizing a 3D-printed esophagus scaffold with a one-hour exposure to ultraviolet light, a 10-minute soak in ethanol, and three washes with PBS.

After the last wash, place the two-layered tubular scaffold into a nonadherent 24-well tissue culture plate, and gently but uniformly add one times 10 to the six adipose-derived human mesenchymal stem cells per milliliter of basement membrane matrix supplemented with growth medium to the inner surface of the scaffold. Use a pulsatile flow bioreactor system to firmly fix the cell-seeded tubular scaffold to the acrylic holder in the culture chamber of the bioreactor, and add 500 milliliters of growth medium to the chamber. Then, apply 1 dyne per centimeter squared of flow-induced shear stress under a humid atmosphere containing 5%carbon dioxide.

After five days, use a LIVE/DEAD Viability Assay Kit to determine the cell responses on the inner surface of the scaffold according to standard protocols, and image the cells by confocal microscopy. Before beginning the surgical procedure, confirm a lack of response to pedal reflex in the anesthetized rat, and place the animal in the supine position on a sterile drape. Use clippers to remove the hair from the surgical site, and disinfect the exposed skin with sequential BETADINE and 70%ethanol scrubs.

For T-tube placement, make a midline skin and muscle incision in the abdomen of the rat, and use a scalpel blade to create a three-millimeter orifice in the anterior gastric wall. Insert the tip of a silicone T-tube into the defect site to fix it to the stomach wall, and carefully suture the tube to the gastric muscle tissue. Suture the abdominal wall with 4-0 VICRYL suture.

Carefully insert the distal end of the implanted T-tube through the subcutaneous tunnel into the back of the neck, and use an angiocatheter to connect a heparin cap to the end of the T-tube to prevent the stomach contents from flowing backwards. When the cap is in place, use 4-0 polyglactin sutures to close all the layers of the abdominal skin, and place the rat into a metabolic cage with monitoring until full recumbency. One week after the T-tube placement, prepare the rat for surgery as demonstrated.

Place the rat under the microscope, and make an anterior median neck incision in the exposed disinfected skin. Separate the strap muscles to expose the tracheoesophageal structure. Isolate the left side of the esophagus from the trachea.

Carefully separate the upper part of the esophagus from the thyroid gland, and use surgical scissors to create a five-millimeter-long full circumferential defect containing all of the layers of the esophagus. Next, insert a 9-0 suture between the right inferoposterior margin of the upper esophagus remnant and scaffold, and continue suturing from right to left between the upper esophagus remnant and the scaffold to create a microanastomosis at the upper end of the distal esophageal defect. Then, anastomose the scaffold in the same manner as the upper margin of the lower esophagus remnant.

When the anastomoses are complete, lay the surrounding thyroid gland flap over the transplanted site to ensure a vascular supply to and stable maintenance of the graft. Then, stitch the subcutaneous muscle and skin tissue with a 4-0 VICRYL suture, and place the rat into a metabolic cage on an infrared warming device with monitoring until full recumbency. After an even application to the inner wall of the scaffold via injection, the human mesenchymal stem cell-embedded basement membrane matrix demonstrates the expected mesenchymal stem cell morphology as assessed by scanning electron microscopy.

After esophageal transplantation into rats with full circumferential esophageal defects as demonstrated, the graft is covered with a thyroid gland flap for stable fixation and vascular supply to the implanted site. The esophageal transplanted rats remain at 340 grams until about nine days after the procedure, at which point the animals rapidly decrease in weight due to various causes, resulting in death by day 15. Although most rats develop non-esophageal obstruction caused by hairballs, overall there is no gross evidence of perforation, anastomosis leakage with fistula, seroma accumulation, abscess formation, or surrounding soft-tissue necrosis in any experimental animal.

Re-epithelialization of the transplantation site can be confirmed by immunofluorescence staining for keratin, and the regenerated collagen layer and the elastin fibers can be clearly observed by Masson's trichrome staining. Regeneration of the esophageal muscle layer is also evidenced by an abundant neovascularization as assessed by desmin immunohistochemical analysis. The success of this procedure relies on the stable maintenance of the T-tube via fixation behind the neck to provide nutrition during the esophageal reconstruction.

To overcome the early mortality typical to this procedure, organoid or early vascularization technology could be applied following the esophageal implantation to aid in the functional esophageal reconstruction.