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Summary

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Abstract

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Introduction

The treatment of esophageal disorders, such as congenital malformations and esophageal carcinomas, can lead to structural segment loss of the esophagus. In most cases, autologous replacement grafts, such as gastric pull-up conduits or colon interpositions, have been performed^1,2. However, these esophageal replacements have a variety of surgical complications and reoperation risks³. Thus, the use of tissue-engineered esophagus scaffolds mimicking the native esophagus can be a promising alternative strategy for ultimately regenerating lost tissues^4,5,6.

Although a tissue-engineered esophagus potentially offers an alternative to the current treatments of esophageal defects, there are significant barriers for its in vivo application. Postoperative anastomotic leakage and necrosis of the implanted esophageal scaffold inevitably lead to a lethal infection of the surrounding aseptic space, such as the mediastinum⁷. Therefore, it is extremely important to prevent food or saliva contamination in the wound and nasogastric tube. Gastrostomy or intravenous nutrition should be considered until primary wound healing is completed. To date, esophageal tissue engineering has been performed in large animal models because large animals can be fed only by intravenous hyperalimentation for 2-4 weeks after implantation of the scaffold⁸. However, such a nonoral feeding model has not been established for early survival after esophageal transplantation in small animals. This is because the animals were extremely active and uncontrollable, so they could not keep the feeding tube in their stomachs for an extended period of time. For this reason, there have been few cases of successful esophageal transplantation in small animals.

In view of the circumstances of esophageal tissue engineering, we designed a two-layer tubular scaffold consisting of electrospun nanofibers (inner layer; Figure 1A) and a 3D-printed strand (outer layer; Figure 1B) including a modified gastrostomy technique. The internal nanofiber is made of PU, a non-degradable polymer, and prevents the leakage of food and saliva. The external 3D printed strands are made of biodegradable polycaprolactone (PCL), which can provide mechanical flexibility and adapt to peristaltic movement. Human adipose-derived mesenchymal stem cells (hAD-MSCs) were seeded on the inner layer of the scaffold to promote re-epithelization. The nanofiber structure can facilitate initial mucosal regeneration by providing a structural extracellular matrix (ECM) environment for cell migration.

We have also increased the survival rate and bioactivity of the inoculated cells through bioreactor cultivation. The implanted scaffold was covered with a thyroid gland flap to enable more stable regeneration of the esophageal mucosa and muscle layer. In this report, we describe protocols for esophageal tissue engineering techniques, including scaffold manufacturing, mesenchymal stem cell-based bioreactor cultivation, a bypass feeding technique with modified gastrostomy, and a modified surgical anastomosis technique for circumferential esophageal reconstruction in a rat model.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC No. 17-0164-S1A0) of the Seoul National University Hospital.

1. Scaffold Manufacturing

NOTE: Two-layered esophageal scaffolds are manufactured by combining electrospinning and 3D printing. The inner membrane of the tubular scaffold was fabricated by electrospinning polyurethane (PU) with rotating stainless steel mandrels as the collectors⁹.

1. For the preparation of tubular PU nanofibers, prepare a 20% (w/v) solution of PU polymer by stirring in N,N-dimethylformamide (DMF) for 8 h at room temperature.
2. Place the PU solution on the syringe with a blunt metal needle (22 G), and electrospin on rotating stainless steel mandrels (diameter = 2 mm) at a distance of 30 cm between the needle tip and the rotating collector.
   NOTE: The power supply is set to a high-voltage direct current of 15 kV potential. The feeding rate of the solution is fixed at 0.5 mL/h using a syringe pump.
3. Make a tubular nanofiber layer on the surface of the mandrel rotating at 3.14 m/s.
4. Dry the PU nanofiber in a vacuum oven at 40 °C overnight to completely remove residual solvent.
   NOTE: The 3D-printed outer wall of the esophageal scaffold is prepared using a rapid prototyping system. The 3D printing equipment consists of a dispenser, nozzle, compression/heat controller, 3-axis conversion stage, and software system.
5. PCL pellets are dissolved at 100 °C in a heating cylinder and then printed on the surface of the nanofibers at high pressure (7 bar) under the control of a bioplotting system. The nozzle size is 300 µm and the strand distance is 700 µm.
6. After removing the two-layered scaffold from the mandrel, sterilize by soaking in 70% ethanol under ultraviolet light.
   NOTE: More detailed characteristics of the scaffold have been reported in previous studies¹⁰.

2. Cell Seeding on the Grafts and Bioreactor Cultivation

NOTE: Human adipose-derived mesenchymal stem cells (hMSCs) purchased from a company were used without modification.

1. Prior to cell transplantation, sterilize the 3D printed esophagus scaffold for 1 h under ultraviolet light, wet it for 10 min with ethanol, and wash it 3x with phosphate-buffered saline (PBS).
2. Culture and expand the hMSCs in growth medium (basal medium/growth supplement). Two-layered tubular scaffolds were transferred into nonadherent 24 well tissue culture plates.
3. To attach the cells to the inner surface of the scaffold, gently add the hMSC suspension at a density of 1 x 10⁶ cells/mL in basement membrane matrix containing the growth medium.
4. Uniformly deposit the basement membrane matrix suspension on the inner surface of the two-layered tubular scaffold.
5. Firmly fix the hMSC-seeded tubular scaffold to the acrylic holder in the culture chamber of the bioreactor using a pulsatile flow bioreactor system.
   NOTE: The custom-designed bioreactor system consists of a pump, bubble trap, flow chamber, pressure gauge, controllable valve, and medium reservoir. When applying shear stress in the culture chamber, allow a resting time of 1-2 min¹¹.
6. Add growth medium to the culture chamber and apply 0.1 dyne/cm² flow-induced shear stress under a humidified atmosphere containing 5% CO₂¹⁰.
   NOTE: The value of flow-induced shear stress was calculated by simulating the peristalsis of the esophageal tissue derived from the human body from previous studies¹⁰.
7. Determine the cell responses on the inner surfaces of the two-layered tubular scaffolds without bioreactor cultivation after 5 days using a LIVE/DEAD Viability Assay Kit according to the manufacturer's instructions. Obtain images via confocal microscopy using the Z-stack tool.
8. On the third day, observe surface morphology of the hMSC-seeded tubular scaffold through a scanning electron microscope (SEM).
   1. Fix the scaffold that was incubated with hMSC with 2.5% glutaraldehyde and OsO4 for 24 h and dehydrate with ethanol.
   2. Coat the fixed hMSCs with platinum using a sputter coater under argon atmospheric conditions and obtain SEM images at an accelerating voltage of 25 kV.

3. Surgical Preparation for Animal Surgery

NOTE: Surgical preparations are applied before both gastrostomy and esophageal transplantation.

1. Set up the sterile surgical instruments: Scalpel blade, Weitlaner retractor, microneedle holder, microsuture forceps, microtissue forceps, microscissors, Mayo-Hegar needle holder, operating scissors, iris scissors, dressing forceps, tissue forceps, splinter forceps, iris forceps, 5 mL syringe (21 G needle), 10 mL syringe (22 G needle), 9-0 polyamide suture, 4-0 polyglactin suture.
2. Anesthetize the animal with an intramuscular injection of tiletamine/zolazepam (50 mg/g dose) and 2% xylazine hydrochloride (2 mg/kg dose).
   NOTE: Adult Sprague-Dawley (SD) rats weighing 398-420 g were used for esophageal transplantation.
3. Before transferring to the surgical drape, check the appropriate anesthetic condition of the animal by pinching the tail with the forceps.
4. Place the animal in a supine position on the sterile drape and use clippers to remove the hair from the neck (for esophageal transplantation) or abdomen (for gastrostomy). Then scrub the surgical site with betadine and 70% ethanol.
5. Prior to incision, subcutaneously inject an analgesic such as buprenorphine (0.05-0.1 mg/kg) for pain relief.

4. Gastrostomy Surgery Using a T-tube in Rats

NOTE: A modified gastrostomy was performed in all experimental animals to allow temporary bypass nonoral tube feeding (n = 5).

1. Have rats fast the day before surgery. Prepare surgery as in section 3.
2. Expose the stomach through a midline incision of the skin and abdominal muscles of the anesthetized rats.
3. Create a 3 mm orifice in the anterior gastric wall with a scalpel blade.
4. Insert the tip of the silicone T-tube into the defect site to fix it to the stomach wall.
5. Suture properly so that the T-tube does not detach from the gastric wall.
6. Take out the distal end of the implanted T-tube through the subcutaneous tunnel into the back of the neck.
7. Insert the heparin cap to the end of the T-tube to prevent the stomach contents from flowing backwards.
   NOTE: Use an angiocatheter to connect the end of the T-tube with the heparin cap.
8. Suture all layers of the abdominal wall and skin using 4-0 polyglactin sutures.
9. Keep all experimental rats separate in a metabolic cage after the gastrostomy has been completed.

5. Esophageal Transplantation

NOTE: The esophageal transplantation of the two-layered tubular scaffold is performed 1 week after the gastrostomy (n = 5). Prior to the transplantation, inoculate hMSCs (cell density: 1 x 10⁶ cells/mL in basement membrane matrix) into the inner wall of each scaffold and incubate for 3 days in the bioreactor system. The surgical procedure is as follows.

1. Remove the neck hair of the model animals and perform standard draping of the surgical site for aseptic surgery.
   NOTE: Create a large shaving area is recommended to maintain asceptic surgery on the animal.
2. After anterior median incision of the neck, separate the strap muscles and expose the tracheoesophageal structure.
3. Bluntly dissect the vagus nerve from the esophagus before resecting the segment, otherwise the animal's breathing is compromised.
4. Under magnification, isolate the left side of the esophagus from the trachea and carefully separate the upper part from the thyroid gland.
5. Create a 5 mm long full circumferential defect containing all layers of the esophagus using surgical scissors.
   NOTE: Prior to esophageal transplantation, cut the prepared scaffolds using surgical scissors to match the length of the transplant site.
6. Under a microscope, perform microanastomosis at both ends of the distal esophageal defect using a 9-0 suture thread. Place the first suture between the right inferoposterior margin of the upper esophagus remnant and scaffold. Continue suturing from right to left between the upper esophagus remnant and the scaffold. Anastomose the scaffold in the same manner as the upper margin of the lower esophagus remnant.
   NOTE: Perform microvascular anastomosis as used in clinical surgery for esophageal transplantation. Work with a microscope for precise, watertight suturing of the implant site.
7. Afterwards, lay the surrounding thyroid gland flap over the transplanted site to ensure stable maintenance of and vascular supply to the grafts.
8. After transplantation, stitch the subcutaneous muscle and skin tissue with a 4-0 vicryl suture.
9. Keep all experimental rats individually in metabolic cages.

6. Post-operative procedures

NOTE: Postoperative procedures are performed after both gastrostomy and esophageal transplantation.

1. After closure of the abdominal wound, put the rats into individual metabolic cages and place the cages on infrared warming devices to prevent hypothermia.
2. Monitor the animals until they achieve and maintain sternal recumbency (i.e., lying upright on chest).
3. To minimize inflammation at the surgical site, administer the antibiotic gentamicin (20 mg/kg) daily to the rats.
4. Begin oral liquid feeding on the third postoperative day until the endpoint of the study. Supply the whole nutrition formula (20.6 g/100 ml [g%] carbohydrate, 3.8 g% protein, 0.2 g% fat) through the heparin cap 3x per day beginning the day after surgery.
5. Check the animals' appearance and body weight daily. Check to manage behavior, such as self-harming incision site or resistance to the tube intake, as well as various surgical complications. When the body weight of the rat models decreases rapidly by 20% or more, perform euthanasia by CO₂ inhalation.

7. Histology and immunohistochemistry

NOTE: For histological analysis, all of the esophageal tissue of the euthanized animals is extracted using surgical scissors. Hematoxylin and eosin staining and Masson's trichrome staining were performed using standard histological techniques. Immunohistochemistry was performed according to the following protocol.

1. Fix the whole esophagus containing the transplanted sites in 4% paraformaldehyde. Create a paraffin block and cut 4 µm thick sections.
2. Deparaffinize the tissue sections and dehydrate them in an ethanol series. Immerse the tissue slides in citrate buffer and heat for 10 min in the microwave. Cool the cells with cold PBS for 20 min. Immerse in 3% hydrogen peroxide for 6 min, and wash with PBS for 10 min.
3. Incubate in 3% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific reactions of tissue sections.
4. Wash 3x with PBS for 5 min. Incubate with primary antibodies against Desmin (diluted to 1:200), keratin 13 (diluted to 1:100), and von Willebrand Factor (vWF; diluted to 1:100) overnight at 4 °C.
5. Wash 3x with PBS for 15 min. Incubate with the appropriate secondary antibody at a concentration of 1:500 for Desmin and Keratin 13 at room temperature. Then, wash the slides twice with PBS for 10 min.
   NOTE: Tissue sections for vWF were incubated using a horseradish peroxidase-conjugated kit (see Table of Materials) and then visualized using 3,3'-diaminobenzidine (DAB).
6. Mount using a glass coverslip and 4',6-diamidino-2-phenolindole (DAPI) containing mounting medium.

Representative Results

Figure 1 shows a schematic diagram of the manufacturing process of the PU-PCL two-layered tubular scaffold. The PU solution was electrospun from an 18 G needle to make a cylindrical internal structure with a thickness of 200 µm. Then, the melted PCL was printed on the outer wall of the PU nanofiber at regular intervals. The surface morphology of the inner and outer walls of the completed tubular scaffold can be seen in the scanning electron microscopy images.

Figure 2 shows the process of inserting a gastrostomy tube in a rat for external nutrient supply (Figure 2A). The T-shaped silicone tube was inserted into the stomach wall and sutured (Figure 2B). The tube was then moved through the subcutaneous tunnel to the back of the neck and connected with a heparin cap (Figure 2C). The tube facilitates the injection of liquid food. It also prohibits the reverse flow of the gastric contents through the tubes.

Figure 3 shows the process of cell inoculation on the scaffold's inner wall, bioreactor cultivation, and esophageal transplantation. The hMSC-embedded basement membrane matrix was applied evenly to the inner wall of the scaffold via injection (Figure 3A). The SEM image shows the morphology of the cell-attached inner surface. Live/dead staining to analyze cell viability on the two-layered tubular scaffold (luminal surface) indicated that most cells were viable, and they spread well on the nanofiber structure in 5 days. The scaffold inoculated with the cells was fixed to the bioreactor, and the shear stress was applied by the pump (Figure 3B). The hMSC-seeded tubular scaffolds, including bioreactor cultivation, were transplanted into rats with full circumferential esophageal defects via microsuture techniques. The graft was covered with a thyroid gland flap for stable fixation and vascular supply of the implanted site (Figure 3C). The weight change of the rats after transplantation was observed until the end of the experiment. Esophageal transplanted rats remained at 340 g until the 9th day, but then rapidly decreased in weight due to various causes (Figure 3D). As a result, most animals died within 15 days.

Figure 4 shows esophageal regeneration after graft implantation. Although most rats developed neoesophageal obstruction caused by hairballs, there was no gross evidence of perforation, anastomosis leakage with fistula, seroma accumulation, abscess formation, or surrounding soft-tissue necrosis in any experimental rat. Re-epithelialization of the transplantation site was confirmed by immunofluorescence staining for keratin 13. The morphology of the collagen layer and the elastin fibers was clearly confirmed at the regeneration site. The presence of abundant elastin and collagen fibers can contribute to better mechanical properties. Regeneration of the esophageal muscle layer was exhibited by desmin immunohistochemistry, and abundant neovascularization was observed at this site.

Figure 1: Schematic illustration of the process used to fabricate 2-layered tubular scaffolds. After fabricating the inner membrane by electrospinning using PU (A), the structural strength of the tubular scaffold was reinforced by adding strands to the outer surface of the membrane using a 3D printing system without solvent (B). The SEM image shows the morphology of the inner and outer layers of the 2-layered tubular scaffold. (Abbreviations: PU = polyurethane). Please click here to view a larger version of this figure.

Figure 2: Gastrostomy. (A) A schematic diagram showing gastrostomy techniques through T-tube insertion in the gastric wall. (B) A puncture hole is made in the middle of the forestomach, and the T-tube tip is inserted into the forestomach. (C) The inlet part of the T-tube is located with the heparin cap in the middle of the occiput. The figure below presents a T-tube gastrostomy apparatus with different components. Please click here to view a larger version of this figure.

Figure 3: Esophageal transplantation. (A) The hMSCs encapsulated in basement membrane matrix were seeded onto the inner layers of the two-layered tubular scaffold. The SEM image shows the morphology of the hMSCs on the inner wall. Viability of the inoculated cells was also confirmed by live-dead staining (green = live cells). The hMSC-seeded scaffolds were immediately incubated in a bioreactor system (B), and then the tissue-engineered esophagi were implanted into the cervical esophagus (C). The implanted site was covered with a thyroid gland flap for stable esophageal reconstruction (arrows). (D) Weight loss studies after esophageal transplantation. Weight loss was determined as absolute change from initial weight of the rats. Please click here to view a larger version of this figure.

Figure 4: Whole histology of the reconstructed esophagus 2 weeks after orthotopic scaffold implantation. Masson's trichrome staining shows the collagen deposition around the implanted sites. Regeneration of the esophageal muscle and mucous layers was confirmed by desmin (green) and keratin 13 (red) immunostaining, respectively. Additionally, neovascularization (arrows) was clearly observed around the regenerated mucosal layer. Please click here to view a larger version of this figure.

Discussion

Existing animal studies on artificial esophagi are still limited by several critical factors. The ideal artificial esophageal scaffold should be biocompatible and have excellent physical properties. It should be able to regenerate the mucosal epithelium in the early postoperative period to prevent anastomotic leakage. Regeneration of the inner circular and outer longitudinal muscle layers is also important for functional peristalsis^12,13.

The mechanical characteristics of the esophagus are essential because the esophagus collapses during respiration and opens during swallowing, with constant exposure to maximal stretching with a recoil phenomenon¹⁴. The implanted scaffold must have these mechanical characteristics as well. The viscoelasticity of the implanted esophagus should be adequate for repetitive ramp-relaxation of the peristaltic movement through the esophagus. Scaffolds that are too weak may rupture or leak and cause severe conditions (e.g., mediastinitis) in the recipient. In contrast, a scaffold that is too stiff could bulge into the esophageal lumen and prevent food passage. Electrospun nanofibers have very favorable physical properties for esophageal reconstruction. The ECM's topographical nature provides an environment favorable for the migration and differentiation of epithelial cells in esophageal layers¹⁵. It also has a nanopore structure that prevents leakage of saliva and various pathogens¹⁶. However, scaffolds made of electrospun nanofibers have limited use due to their soft mechanical properties. To solve this problem, we improved their mechanical strength using 3D printing technology. The 3D printed strand on the outer layer of the nanofiber has a width of 780 µm, and the inner pore structure is quite wide. It provides physical support for esophageal interventions rather than guiding the regeneration of the surrounding tissue.

In this study, circumferential esophageal defects were fully healed in the bioreactor cultured grafts for up to 2 weeks, but all experimental rats died within 15 days after surgery. Most deaths were caused by peritonitis and malnutrition caused by food and saliva leaks proximal to the anastomosis site. All animals freely consumed a liquid diet for up to a week, but as the wound healing progressed, unintended mechanical obstruction occurred in the reconstructed esophagus due to hairball swallowing. This phenomenon has been shown to cause complete digestive disorder within the implanted non-dynamic scaffolds. There are several options to overcome these technical issues. First, the development of a highly elastic esophageal implant that can mimic esophageal peristalsis. Second, animal studies using hairless rats to prevent hair swallowing. Third, the biliary stent can be applied simultaneously with the scaffold to minimize implant collapse and anastomosis damage. In addition, the application of microvascular anastomosis to esophageal scaffold implantation is important to completely prevent the leakage of saliva. The conventional suture technique using the naked eyes is extremely difficult to make watertight in rat models.

A reliable vascular vehicle is essential for nutrients, growth factors, and oxygen supply in the early stages of regeneration. The thyroid gland is vascular tissue located near the esophagus. We used the thyroid gland flap after circumferential esophagectomy because of its easy accessibility in the rat model. In conclusion, we propose various preclinical techniques to overcome the difficulties of esophageal reconstruction in the rat model. This study presents a good alternative to overcome the limitations of conventional small animal esophagus transplantation.

Materials

Metabolic cage	TEUNGDO BIO & PLANT	JD-C-66
Zoletil (50 mg/g dose)	Virbac	1000000188
0.25% Trypsin-EDTA	Gibco	25200-056
1 mL Syringe	BD	309659
2% xylazine hydrochloride (Rumpun)	Byely	Q-0615-035
4% paraformaldehyde	BIOSOLUTION	BP031
4-0 Vicryl	ETHICON	W9443
9-0 Vicryl	ETHICON	W2813
Antibiotic gentamicin (Septopal).	Septopal	0409-1207-03
Bovine Serum Albumin (BSA)	Sigma	5470
Citrate Buffer, ph6.0, 10X	Sigma	C9999
DAB PEROXIDASE SUBSTRATE KIT	VECTOR	SK4100
Desmin	Santa Cruz	sc-23879
Elastic stain kit	ScyTeK	ETS-1
Ethanol	Merck	100983
Ethanol	Merck	64-17-5
Fetal Bovine Serun (FBS)	Gibco	16000-044
Glutaraldehyde	Sigma	354400
Goat anti-Mouse IgG (H+L) Secondary Antibody	ThermoFisher	A-11001
Heparin cap	Hyupsung Medical	HS-T-05
hMSC (STEMPRO) / growth medium (MesenPRO RSTM)	Invitrogen	R7788-110
Horseradish peroxidase-conjugated kit (Vectastain)	VECTOR	PK7800
Hydrogen peroxide	JUNSEI	7722-84-1
Keratin13	Novus	NBP1-97797
LIVE/DEAD Viability Assay Kit	Molecular Probes	L3224
Matrigel	Corning	354262
N,N-dimethylformamide (DMF)	Sigma	227056
Nonadherent 24-well tissue culture plates.	Corning	3738
OsO4	Sigma	O5500
Petri dish	Eppendorf	3072115
Phosphate-buffered saline (PBS)	Gibco	10010-023
Phosphate-buffered saline (PBS), 10X	BIOSOLUTION	BP007a
Polycaprolactone (PCL) polymer	Sigma	440744
Polyurethane (PU+A2:A24) polymer	Lubrizol	2363-80AE
Power Supply	NanoNC	HV100
ProLong Gold antifade reagent with DAPI	Invitrogen	P36931
Rumpun	Bayer	Q-0615-035
Silicone T-tube	Sewoon Medical	2206-005
Terramycin Eye Ointment	Pfizer Pharmaceutical Korea	W01890011
Tiletamine/Zolazepam (Zoletil)	Virbac Laboratories	Q-0042-058
Trichrome stain kit	ScyTeK	TRM-1
von Willebrand Factor (vWF)	Santa Cruz	sc 14014