Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

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,

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....

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........

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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