We would like to acknowledge Robert Strouse and the Research Information Solutions &#38; Innovations division at Nationwide Children&#39;s Hospital for their support in graphic design. This work was supported by a grant from the NIH (NHLBI K08HL138460).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at Nationwide Children&#39;s Hospital.
1. Scaffold Manufacturing
<ol>
	<li>Prepare a polymer nanofiber precursor solution by: 1) dissolving 8 wt% PET in 1,1,1,3,3,3-hexafluoroisopropanol and heating the solution to 60 &#176;C and by 2) dissolving 3 wt% PU in 1,1,1,3,3,3-hexafluoroisopropanol at room temperature.</li>
	<li>Once cooled, combine the solutions to create a final polymer mixtur...

<ol>
	<li>Macchiarini, P., 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=Clinical+transplantation+of+a+tissue-engineered+airway.">Clinical transplantation of a tissue-engineered airway.</a> The Lancet. 372 (9655), 2023-2030 (2008).</li><li>Jungebluth, P., 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=Tracheobronchial+transplantation+with+a+stem-cell-seeded+bioartificial+nanocomposite:+A+proof-of-concept+study.">Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: A proof-of-concept study.</a> The Lancet. 378 (9808), 1997-2004 (2011).</li><li>Elliott, M. 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=Stem-cell-based,+tissue+engineered+tracheal+replacement+in+a+child:+A+2-year+follow-up+study.">Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study.</a> The Lancet. 380 (9846), 994-1000 (2012).</li><li>Chiang, T., Pepper, V., Best, C., Onwuka, E., Breuer, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Translation+of+Tissue+Engineered+Trachea+Grafts.">Clinical Translation of Tissue Engineered Trachea Grafts.</a> Annals of Otology, Rhinology and Laryngology. 125 (11), 873-885 (2016).</li><li>Clark, E. 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=Effect+of+cell+seeding+on+neotissue+formation+in+a+tissue+engineered+trachea.">Effect of cell seeding on neotissue formation in a tissue engineered trachea.</a> Journal of Pediatric Surgery. 51 (1), 49-55 (2016).</li><li>Cole, B. B., Smith, R. W., Jenkins, K. M., Graham, B. B., Reynolds, P. R., Reynolds, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+basal+cells:+A+facultative+progenitor+cell+pool.">Tracheal basal cells: A facultative progenitor cell pool.</a> American Journal of Pathology. 177 (1), 362-376 (2010).</li><li>Onwuka, 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=The+role+of+myeloid+cell-derived+PDGF-B+in+neotissue+formation+in+a+tissue-engineered+vascular+graft.">The role of myeloid cell-derived PDGF-B in neotissue formation in a tissue-engineered vascular graft.</a> Regenerative Medicine. 12 (3), 249-261 (2017).</li><li>Grimmer, J. 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=Tracheal+reconstruction+using+tissue-engineered+cartilage.">Tracheal reconstruction using tissue-engineered cartilage.</a> Archives of Otolaryngology - Head and Neck Surgery. 130 (10), 1191-1196 (2004).</li><li>Wood, M. W., Murphy, S. V., Feng, X., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+reconstruction+in+a+canine+model.">Tracheal reconstruction in a canine model.</a> Otolaryngology - Head and Neck Surgery (United States). 150 (3), 428-433 (2014).</li><li>Haag, 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=Biomechanical+and+angiogenic+properties+of+tissue-engineered+rat+trachea+using+genipin+cross-linked+decellularized+tissue.">Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue.</a> Biomaterials. 33 (3), 780-789 (2012).</li><li>Best, C. 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=Designing+a+tissue-engineered+tracheal+scaffold+for+preclinical+evaluation.">Designing a tissue-engineered tracheal scaffold for preclinical evaluation.</a> International Journal of Pediatric Otorhinolaryngology. 104, 155-160 (2018).</li></ol>

The authors declare no competing financial interests.

Development of a mouse model for tissue engineered tracheas is essential in understanding the factors that have limited clinical translation of the TETGs; namely graft collapse, stenosis and delayed epithelialization4. A few factors that contribute to these limitations include selection of graft material, the manufacturing process, scaffold design and cell seeding protocols. This model allows for faster evaluation of these factors in order to understand the cellular and molecular mechanisms affect...

Long-segment tracheal defects can present as rare congenital conditions such as complete tracheal rings and tracheal agenesis, as well as trauma, malignancy, and infection. When exceeding 6 cm in adults or 30% of the tracheal length in children, these defects cannot be treated by surgical reconstruction. Attempts to replace the airway with autologous tissue, cadaveric transplants, and artificial constructs have been plagued by chronic infection, granulation, mechanical failure, and stenosis.
Tissue engineered tracheal grafts (TETGs) can potentially address these problems while avoiding the need for life-long immunosuppression. In the last decade, TETGs have been tested in animal models and utilized clinically in rare instances of compassionate use1,2,3. In both clinical and large animal studies, post-operative recovery from tissue engineered airway replacement required numerous interventions to combat stenosis (defined as &#62;50% luminal narrowing) and maintain airway patency. Additional TETG work has sought to reduce this stenosis through evaluating the role of cell seeding choice, vascularization and scaffold design. Cell seeding choices and scaffold design aimed at restoring native trachea structure/function have mainly been focused on respiratory epithelial cells and chondrocytes seeded on various resorbable, non-resorbable and decellularized scaffolds. As vascularization likely plays an major role in the development of stenosis, other groups have focused on optimizing in vitro or heterotopic models to expedite revascularization or neoangiogenesis4. Nonetheless, achieving successful vascularization while also maintaining a mechanically competent and functional TETG remains a challenge. Despite recent advances, minimizing stenosis remains a critical obstacle to clinical translation.
To investigate this histopathological response to TETG implantation in vivo, we developed an ovine model of tissue engineered tracheal replacement. The graft was composed of a mixed polyethylene terephthalate (PET) and polyurethane (PU) electrospun scaffold seeded with bone marrow-derived mononuclear cells (BM-MNCs). In this small cohort, we demonstrated that seeded autologous BM-MNCs accelerated re-epithelialization and delayed stenosis5. Although seeding with autologous BM-MNCs improved survival, the cellular mechanism by which BM-MNCs modulate the formation of functional neotissue remains unclear.
Investigation on the cellular level required development of a murine model of tissue engineered tracheal replacement. Similar to the ovine study, we utilized a PET:PU electrospun scaffold seeded with BM-MNCs. Consistent with the ovine model, TETG stenosis developed over the course of the first two weeks following implantation1,2,3,5. This suggested that the murine model recapitulated the pathology observed previously, enabling us to further interrogate the cellular mechanisms underlying airway stenosis.
In this report, we detail our protocol for tissue engineered tracheal replacement in the mouse model including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation (Figure 1, Figure 2).

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">0.9% Sodium chloride injection</td>
			<td style="width:119px;">APP Pharmaceuticals</td>
			<td style="width:119px;">NDC 63323-186-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">10cc serological pipet</td>
			<td style="width:119px;">Falcon</td>
			<td style="width:119px;">357551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">18G 1.5in. Needle</td>
			<td style="width:119px;">BD</td>
			<td style="width:119px;">305190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">1mL Syringe</td>
			<td style="width:119px;">BD</td>
			<td style="width:119px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">24-well plate</td>
			<td style="width:119px;">Corning</td>
			<td style="width:119px;">3526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">25cc serological pipet</td>
			<td style="width:119px;">Falcon</td>
			<td style="width:119px;">356535</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">25G 1in. Needle</td>
			<td style="width:119px;">BD</td>
			<td style="width:119px;">305125</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">50cc tube</td>
			<td style="width:119px;">BD</td>
			<td style="width:119px;">352070</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Alcohol prep pads</td>
			<td style="width:119px;">Fisher Healthcare</td>
			<td style="width:119px;">NDC 69250-661-02</td>
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			<td height="42" style="height:42px;width:423px;">Baytril (enrofloxacin) solution</td>
			<td style="width:119px;">Bayer Healthcare, LLC</td>
			<td style="width:119px;">NDC 0859-2267-01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Black polyamide monofilament suture, 9-0</td>
			<td style="width:119px;">AROSurgical Instruments Corporation</td>
			<td style="width:119px;">T05A09N10-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">C57BL/6, female</td>
			<td style="width:119px;">Jackson laboratories</td>
			<td style="width:119px;">664</td>
			<td style="width:167px;">6-8 weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Citrate Buffer pH 6.0 20x concentrate</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">5000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Colibri retractors</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">17000-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cotton tipped applicators</td>
			<td style="width:119px;">Fisher scientific</td>
			<td style="width:119px;">23-400-118</td>
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			<td height="21" style="height:21px;width:423px;">Cytokeratin 14 Monoclonal Antibody</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">MA5-11599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dumont #5 Forceps</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">11251-20</td>
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			<td height="21" style="height:21px;width:423px;">Dumont #5/45 forceps</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">11251-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dumont #7 - Fine Forceps</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">F4/80 Rat anti-mouse antibody</td>
			<td style="width:119px;">Bio-Rad</td>
			<td style="width:119px;">MCA497R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ficoll</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">10831-100mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Fine scissors- Sharp-blunt</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Fisherbrand Premium Cover Glasses</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">12-548-5M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Fluoroshield Mounting Media with DAPI</td>
			<td style="width:119px;">Abcam</td>
			<td style="width:119px;">ab104139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Goat-anti mouse IgG Secondary Antibody Alexa Fluor 594</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">A-11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Goat-anti Rabbit IgG Secondary Antibody Alexa Fluor 594</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">A-11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Goat-anti Rat IgG Secondary Antibody Alexa Fluor 647</td>
			<td style="width:119px;">ThermoFisher</td>
			<td style="width:119px;">A-21247</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Ibuprofen</td>
			<td style="width:119px;">Precision Dose, Inc</td>
			<td style="width:119px;">NDC 68094-494-59</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:423px;">Iodine prep pads</td>
			<td style="width:119px;">Professional disposables international, Inc.</td>
			<td style="width:119px;">NDC 10819-3883-1</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Keratin 5 Polyclonal Antibody, Purified</td>
			<td style="width:119px;">BioLegend</td>
			<td style="width:119px;">905501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ketamine hydrochloride injection</td>
			<td style="width:119px;">Hospira Inc.</td>
			<td style="width:119px;">NDC 0409-2053</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Micro-Adson forceps</td>
			<td style="width:119px;">F.S.T</td>
			<td style="width:119px;">11018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:119px;">M80</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Non-woven sponges</td>
			<td style="width:119px;">Covidien</td>
			<td style="width:119px;">441401</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:423px;">Opthalmic ointment</td>
			<td style="width:119px;">Dechra Veterinary products</td>
			<td style="width:119px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">PBS</td>
			<td style="width:119px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">PET/PU (Polyethylene terephthalate &#38; Polyurethane) scaffolds</td>
			<td style="width:119px;">Nanofiber solutions</td>
			<td style="width:119px;"></td>
			<td>Custom ordered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Petri dish</td>
			<td style="width:119px;">BD</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">RPMI 1640 Medium</td>
			<td style="width:119px;">Gibco</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">TISH Needle Holder/Forceps</td>
			<td style="width:119px;">Micrins</td>
			<td style="width:119px;">MI1540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Trimmer</td>
			<td style="width:119px;">Wahl</td>
			<td style="width:119px;">9854-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas-T&#252;bingen Spring Scissors</td>
			<td>F.S.T</td>
			<td>15008-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Warm water recirculator</td>
			<td style="width:119px;">Gaymar</td>
			<td style="width:119px;">TP-700</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Xylazine sterile solution</td>
			<td style="width:119px;">Akorn animal health</td>
			<td style="width:119px;">NDC 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 illustrates a schematic of TETG seeding and implantation. Bone marrow was harvested from C57BL/6 mice and cultured in vitro. BM-MNCs were isolated by density centrifugation and seeded onto the TETG. Seeded TETGs were implanted into a syngeneic C57BL/6 recipient mouse.
Figure 2 is an overview of the PET:PU TETG scaffold manufacturing process. PET:PU sol...

Watch this Scientific Journal Video about Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model at JoVE.com

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

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 ...

Clinical applications of tissue-engineered trachea have been limited due to graft stenosis and delayed epithelialization. A mouse model of orthotopic tracheal replacement permits the study of the cellular mechanisms driving regeneration. Long-segment defects of the airway can exceed what current therapies are able to cure.The tissue-engineered trachea offers the potential to create a replacement organ for these conditions. This model can be applied to animals with transgenic and lineage tracing properties. We can then modulate the host factors contributing to graft regeneration.One of the key challenges in producing the scaffolds will be measuring and determining that the electrospinning parameters are the same each time. So every time you produce a scaffold, you need to measure the tip-to-collector distance, the humidity, and the temperature to ensure the scaffolds are the same each time. Critical to the success of this model is refinement of the microsurgical techniques and maintenance of the correct plane of anesthesia to permit spontaneous ventilation throughout the procedure.Since the host trachea has an internal diameter of around one millimeter, graft and native tissue handling and suture placement are important and best demonstrated visually. Begin by dissolving eight weight percent polyethylene terephthalate, or PET, in hexafluoroisopropanol and heating the resulting solution to 60 degrees Celsius. While the solution is being warmed, dissolve three weight percent polyurethane, or PU, in hexafluoroisopropanol, combining the solutions once the PET solution has cooled.Load a 60-milliliter syringe equipped with a 20-gauge blunt-tip needle with the polymer mixture. Use the syringe and a high-voltage DC power supply set to positive 14 kilovolts on the needle, another high-voltage DC power supply set to minus three kilovolts, a five-milliliter-per-hour flow rate, and a 20-centimeter tip-to-substrate distance to electrospin the polymer onto a one-millimeter diameter stainless steel rod being turned at 350 rotations per minute. Continue electrospinning until a 300-micrometer scaffold wall thickness is achieved.Then, slide the scaffold off the rod, place the scaffold under vacuum overnight to remove any residual solvent, and sterilize the scaffold with an ultraviolet light dosage of 350 millijoules per centimeter squared for about 15 minutes. Under sterile conditions, remove the skin from the hind limbs of a six to eight-week-old, female, C57-Black/6 mouse with fine scissors and micro-Adson forceps, and use Dumont number five forceps and Dumont number 5/45 forceps to remove the fascia and the tendons. Separate the bones to allow each of them to be cut on both ends, and use a five-milliliter syringe equipped with a 25-gauge needle to flush the bone marrow into a Petri dish containing 30 milliliters of RPMI medium.When all of the bones have been flushed, filter the bone marrow through a 70-micrometer nylon cell strainer into a 50-milliliter conical tube, and transfer the tube to a biosafety cabinet. Gently add the filtered mononuclear cell solution down the side of a 15-milliliter tube containing five milliliters of polysucrose and sodium diatrizoate without mixing the layers, and separate the plasma from the white and red blood cells by density gradient separation. At the end of the centrifugation, collect the middle clear layer consisting of the bone marrow mononuclear cells, and wash the bone marrow mononuclear cells at a one-to-one ratio in PBS by centrifugation.Resuspend the pellet in five milliliters of PBS for a second wash, followed by resuspension in 10 milliliters of fresh RPMI medium for counting. Then, collect the cells with an additional centrifugation, and dilute the cells to a one times 10 to the seven cells per five microliters of fresh RPMI concentration. Before seeding the scaffold, cut the polymer to a five-millimeter length as necessary, and wet the scaffold with five microliters of RPMI medium for five minutes.At the end of the incubation, remove the excess medium, and load the scaffold lumen with five microliters of the bone marrow mononuclear cell solution for 10 minutes. Then, insert a 21-gauge needle through the lumen of the scaffold, and place the graft in one milliliter of RPMI medium overnight in a 37-degree Celsius incubator. After induction of general anesthesia, confirm a lack of response to toe pinch in the recipient animal, and apply ointment to the animal's eyes.Clip the hair on the surgical site from chin to clavicles, and place the animal on a surgical pad in a dorsal recumbent position. Using aseptic technique, disinfect the surgical site with a sequential povidone-iodine, 70%ethanol, povidone-iodine wipe series, and move the mouse under a dissecting microscope with the head at 12 o'clock. Use fine scissors and micro-Adson forceps to make a midline incision from the clavicles to the hyoid bone, and use Dumont number five and number seven fine forceps and a sterile cotton swab to clean the fascia before inserting a self-retaining Colibri retractor into the incision.Use the forceps to open the strap muscles to expose the thyroid cartilage, cricoid cartilage, and trachea, and bluntly separate the trachea from the recurrent laryngeal nerves running parallel on either side of the tissue, followed by circumferential separation of the trachea from the esophagus. Using a 20-gauge needle and surgical marker, stain the anterior portion of the trachea. Make an incision below the third tracheal cartilage ring using a pair of Vannas-Tubingen spring scissors and a pair of Dumont number seven fine forceps to resect a three-ring section of trachea.Holding the trachea with the fine curved forceps, use a sterile 9-0 nylon suture to secure the distal end of the trachea to the sternum to create a temporary tracheostomy, and use the 20-gauge needle and surgical marker to stain the anterior portion of the graft. To implant the graft, use a 9-0 sterile nylon suture, Dumont number seven fine forceps, and a needle holder to insert the proximal-posterior, proximal-lateral, and proximal-anterior stitches, and turn the animal 180 degrees. When the mouse is in position, release the temporary tracheostomy, and make a lateral incision five millimeters away from the anterior portion of the graft to facilitate the distal anastomosis.To complete the distal anastomosis, place the sutures in a similar fashion to the proximal anastomosis, and re-approximate the graft position and strap muscles. Then, close the incision with 9-0 sterile nylon sutures in a running pattern, and place the animal alone in a recovery cage placed on a heating pad with monitoring until full recovery. Two weeks after implantation, a few basal epithelial cells can be observed by basal cell keratin five and 14 staining in tissue-engineered tracheal graft tissue sections.Basal cells are also detected on the luminal surface of the graft at seven days after implantation. Upon explantation and histological analysis, graft stenosis has been identified as the main contributing factor in graft-implanted mice demonstrating signs of respiratory distress and stridor. Note that telescoping of the graft and native trachea is a common finding as a result of the surgical technique.Staining for F4/80 reveals host macrophages within the stenotic region of the graft in conjunction with the presence of the basal epithelial cells. Avoid manipulation of the recurrent laryngeal nerves, and ensure that the proximal and distal sutures are in place to allow an airtight seal at the anastomosis. As this protocol deals with bone marrow mononuclear cells derived from biosafety level one agent mice, it is important to wear appropriate personal protective equipment to avoid contamination.

seeding-implantation-biosynthetic-tissue-engineered-tracheal-graft

Research

JoVE Journal

Bioengineering

7.1K Views.  Nationwide Children's Hospital. Clinical applications of tissue-engineered trachea have been limited due to graft stenosis and delayed epithelialization. A mouse model of orthotopic tracheal replacement permits the study of the cellular mechanisms driving regeneration. Long-segment defects of the airway can exceed what current therapies are able to cure.The tissue-engineered trachea offers the potential to create a replacement organ for these conditions. This model can be applied to animals with transgenic and lineag...