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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(Baltimore). 96 (21), e6942 (2017).</li><li>Luc, G., 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=Decellularized+and+matured+esophageal+scaffold+for+circumferential+esophagus+replacement:+Proof+of+concept+in+a+pig+model.">Decellularized and matured esophageal scaffold for circumferential esophagus replacement: Proof of concept in a pig model.</a> Biomaterials. 175, 1-18 (2018).</li><li>Wang, F., Maeda, Y., Zachar, V., Ansari, T., Emmersen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+oesophageal+muscle+layer+from+oesophagus+acellular+matrix+scaffold+using+adipose-derived+stem+cells.">Regeneration of the oesophageal muscle layer from oesophagus acellular matrix scaffold using adipose-derived stem cells.</a> Biochemical and Biophysical Research Communications. 503 (1), 271-277 (2018).</li><li>La Francesca, S., 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=Long-term+regeneration+and+remodeling+of+the+pig+esophagus+after+circumferential+resection+using+a+retrievable+synthetic+scaffold+carrying+autologous+cells.">Long-term regeneration and remodeling of the pig esophagus after circumferential resection using a retrievable synthetic scaffold carrying autologous cells.</a> Scientific Reports. 8 (1), 4123 (2018).</li><li>Ponten, J. E., 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=Early+severe+mediastinal+bleeding+after+esophagectomy:+a+potentially+lethal+complication.">Early severe mediastinal bleeding after esophagectomy: a potentially lethal complication.</a> Journal of Thoracic Disease. 5 (2), E58-E60 (2013).</li><li>Catry, J., 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=Circumferential+Esophageal+Replacement+by+a+Tissue-engineered+Substitute+Using+Mesenchymal+Stem+Cells:+An+Experimental+Study+in+Mini+Pigs.">Circumferential Esophageal Replacement by a Tissue-engineered Substitute Using Mesenchymal Stem Cells: An Experimental Study in Mini Pigs.</a> Cell Transplant. 26 (12), 1831-1839 (2017).</li><li>Lee, S. J., 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=Characterization+and+preparation+of+bio-tubular+scaffolds+for+fabricating+artificial+vascular+grafts+by+combining+electrospinning+and+a+3D+printing+system.">Characterization and preparation of bio-tubular scaffolds for fabricating artificial vascular grafts by combining electrospinning and a 3D printing system.</a> Physical Chemistry Chemical Physics. 17 (5), 2996-2999 (2015).</li><li>Kim, I. G., 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=Tissue-Engineered+Esophagus+via+Bioreactor+Cultivation+for+Circumferential+Esophageal+Reconstruction.">Tissue-Engineered Esophagus via Bioreactor Cultivation for Circumferential Esophageal Reconstruction.</a> Tissue Engineering Part A. , (2019).</li><li>Wu, Y., 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=Combinational+effects+of+mechanical+forces+and+substrate+surface+characteristics+on+esophageal+epithelial+differentiation.">Combinational effects of mechanical forces and substrate surface characteristics on esophageal epithelial differentiation.</a> Journal of Biomedical Materials Research A. 107, 552-560 (2019).</li><li>Jensen, T., 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=Polyurethane+scaffolds+seeded+with+autologous+cells+can+regenerate+long+esophageal+gaps:+An+esophageal+atresia+treatment+model.">Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model.</a> Journal of Pediatric Surgery. 3468 (18), 30685-30687 (2018).</li><li>Nakase, Y., 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=Intrathoracic+esophageal+replacement+by+in+situ+tissue-engineered+esophagus.">Intrathoracic esophageal replacement by in situ tissue-engineered esophagus.</a> Journal of Thoracic and Cardiovascular Surgery. 136 (4), 850-859 (2008).</li><li>Kwiatek, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCL+and+PCL-gelatin+nanofibers+as+esophageal+tissue+scaffolds:+optimization,+characterization+and+cell-matrix+interactions.">PCL and PCL-gelatin nanofibers as esophageal tissue scaffolds: optimization, characterization and cell-matrix interactions.</a> Journal of Biomedical Nanotechnology. 9 (9), 1540-1555 (2013).</li></ol>

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.

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Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

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

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 animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

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

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

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. 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 ...

Synthesis of Thermogelling Poly(N-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 the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

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

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

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

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 mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

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

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

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

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

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

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

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 replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

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

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.