Name: tissue-engineered graft for circumferential esophageal reconstruction in rats
Uploaded: 2020-02-10T12:00:00
Description: Watch this Scientific Journal Video about Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats at JoVE.com

This research was supported by the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI16C0362) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1C1B2011132). The biospecimens and data used in this study were provided by the Biobank of Seoul National University Hospital, a member of Korea Biobank Network.

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.</...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+esophagus+in+eosinophilic+esophagitis.">Mechanical properties of the esophagus in eosinophilic esophagitis.</a> Gastroenterology. 140 (1), 82-90 (2011).</li><li>Anjum, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocomposite+nanofiber+matrices+to+support+ECM+remodeling+by+human+dermal+progenitors+and+enhanced+wound+closure.">Biocomposite nanofiber matrices to support ECM remodeling by human dermal progenitors and enhanced wound closure.</a> Scientific Reports. 7 (1), 10291 (2017).</li><li>Kuppan, P., Sethuraman, S., Krishnan, U. 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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 peristalsis12,13.
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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 performed1,2. 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 tissues4,5,6.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Metabolic cage</td>
			<td>TEUNGDO BIO &#38; PLANT</td>
			<td>JD-C-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil (50 mg/g dose)</td>
			<td>Virbac</td>
			<td>1000000188</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>2% xylazine hydrochloride (Rumpun)</td>
			<td>Byely</td>
			<td>Q-0615-035</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>BIOSOLUTION</td>
			<td>BP031</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 Vicryl</td>
			<td>ETHICON</td>
			<td>W9443</td>
			<td></td>
		</tr>
		<tr>
			<td>9-0 Vicryl</td>
			<td>ETHICON</td>
			<td>W2813</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic gentamicin (Septopal).</td>
			<td>Septopal</td>
			<td>0409-1207-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate Buffer, ph6.0, 10X</td>
			<td>Sigma</td>
			<td>C9999</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB PEROXIDASE SUBSTRATE KIT</td>
			<td>VECTOR</td>
			<td>SK4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmin</td>
			<td>Santa Cruz</td>
			<td>sc-23879</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic stain kit</td>
			<td>ScyTeK</td>
			<td>ETS-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serun (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>354400</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody</td>
			<td>ThermoFisher</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin cap</td>
			<td>Hyupsung Medical</td>
			<td>HS-T-05</td>
			<td></td>
		</tr>
		<tr>
			<td>hMSC (STEMPRO) / growth medium 
			(MesenPRO RSTM)</td>
			<td>Invitrogen</td>
			<td>R7788-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase-conjugated kit (Vectastain)</td>
			<td>VECTOR</td>
			<td>PK7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>JUNSEI</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin13</td>
			<td>Novus</td>
			<td>NBP1-97797</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability Assay Kit</td>
			<td>Molecular Probes</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354262</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide (DMF)</td>
			<td>Sigma</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonadherent 
			24-well tissue culture plates.</td>
			<td>Corning</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>Sigma</td>
			<td>O5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Eppendorf</td>
			<td>3072115</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), 10X</td>
			<td>BIOSOLUTION</td>
			<td>BP007a</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL) polymer</td>
			<td>Sigma</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyurethane (PU+A2:A24) polymer</td>
			<td>Lubrizol</td>
			<td>2363-80AE</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply</td>
			<td>NanoNC</td>
			<td>HV100</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Rumpun</td>
			<td>Bayer</td>
			<td>Q-0615-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone T-tube</td>
			<td>Sewoon Medical</td>
			<td>2206-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Terramycin Eye Ointment</td>
			<td>Pfizer Pharmaceutical Korea</td>
			<td>W01890011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine/Zolazepam (Zoletil)</td>
			<td>Virbac Laboratories</td>
			<td>Q-0042-058</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichrome stain kit</td>
			<td>ScyTeK</td>
			<td>TRM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>von Willebrand Factor (vWF)</td>
			<td>Santa Cruz</td>
			<td>sc 14014</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue-engineered graft for circumferential esophageal reconstruction in rats

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.

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 &#181;m. 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 im...

Watch this Scientific Journal Video about Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats at JoVE.com

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

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.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

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.

Research

JoVE Journal

Bioengineering

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Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

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The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...