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>
	<li>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.</li>
	<li>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.</li>
	<li>Make a tubular nanofiber layer on the surface of the mandrel rotating at 3.14 m/s.</li>
	<li>Dry the PU nanofiber in a vacuum oven at 40 &#176;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.</li>
	<li>PCL pellets are dissolved at 100 &#176;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 &#181;m and the strand distance is 700 &#181;m.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Uniformly deposit the basement membrane matrix suspension on the inner surface of the two-layered tubular scaffold.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s instructions. Obtain images via confocal microscopy using the Z-stack tool.</li>
	<li>On the third day, observe surface morphology of the hMSC-seeded tubular scaffold through a scanning electron microscope (SEM).
	<ol>
		<li>Fix the scaffold that was incubated with hMSC with 2.5% glutaraldehyde and OsO4 for 24 h and dehydrate with ethanol.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Surgical Preparation for Animal Surgery
NOTE: Surgical preparations are applied before both gastrostomy and esophageal transplantation.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Before transferring to the surgical drape, check the appropriate anesthetic condition of the animal by pinching the tail with the forceps.</li>
	<li>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.</li>
	<li>Prior to incision, subcutaneously inject an analgesic such as buprenorphine (0.05-0.1 mg/kg) for pain relief.</li>
</ol>
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).
<ol>
	<li>Have rats fast the day before surgery. Prepare surgery as in section 3.</li>
	<li>Expose the stomach through a midline incision of the skin and abdominal muscles of the anesthetized rats.</li>
	<li>Create a 3 mm orifice in the anterior gastric wall with a scalpel blade.</li>
	<li>Insert the tip of the silicone T-tube into the defect site to fix it to the stomach wall.</li>
	<li>Suture properly so that the T-tube does not detach from the gastric wall.</li>
	<li>Take out the distal end of the implanted T-tube through the subcutaneous tunnel into the back of the neck.</li>
	<li>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.</li>
	<li>Suture all layers of the abdominal wall and skin using 4-0 polyglactin sutures.</li>
	<li>Keep all experimental rats separate in a metabolic cage after the gastrostomy has been completed.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>After anterior median incision of the neck, separate the strap muscles and expose the tracheoesophageal structure.</li>
	<li>Bluntly dissect the vagus nerve from the esophagus before resecting the segment, otherwise the animal&#39;s breathing is compromised.</li>
	<li>Under magnification, isolate the left side of the esophagus from the trachea and carefully separate the upper part from the thyroid gland.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Afterwards, lay the surrounding thyroid gland flap over the transplanted site to ensure stable maintenance of and vascular supply to the grafts.</li>
	<li>After transplantation, stitch the subcutaneous muscle and skin tissue with a 4-0 vicryl suture.</li>
	<li>Keep all experimental rats individually in metabolic cages.</li>
</ol>
6. Post-operative procedures
NOTE: Postoperative procedures are performed after both gastrostomy and esophageal transplantation.
<ol>
	<li>After closure of the abdominal wound, put the rats into individual metabolic cages and place the cages on infrared warming devices to prevent hypothermia.</li>
	<li>Monitor the animals until they achieve and maintain sternal recumbency (i.e., lying upright on chest).</li>
	<li>To minimize inflammation at the surgical site, administer the antibiotic gentamicin (20 mg/kg) daily to the rats.</li>
	<li>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.</li>
	<li>Check the animals&#39; 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.</li>
</ol>
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&#39;s trichrome staining were performed using standard histological techniques. Immunohistochemistry was performed according to the following protocol.
<ol>
	<li>Fix the whole esophagus containing the transplanted sites in 4% paraformaldehyde. Create a paraffin block and cut 4 &#181;m thick sections.</li>
	<li>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.</li>
	<li>Incubate in 3% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific reactions of tissue sections.</li>
	<li>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 &#176;C.</li>
	<li>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&#39;-diaminobenzidine (DAB).</li>
	<li>Mount using a glass coverslip and 4&#39;,6-diamidino-2-phenolindole (DAPI) containing mounting medium.</li>
</ol>

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The bioreactor system designed for this study has been commercialized (model number: ACBF-100).

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.
<p class="jove_conte...

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><tbody><tr><td>Metabolic cage</td><td>TEUNGDO BIO &amp;amp; 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&lt;br/&gt; (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&lt;br/&gt; 24-well tissue culture plates.</td><td>Corning</td><td>3738</td><td></td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</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...

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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 ...

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Research

JoVE Journal

Bioengineering

7.1K Views.  Seoul National University, College of Medicine. 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.

Video: Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Medicine

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biology

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. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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 esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

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

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

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

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...