Name: low-dose gamma radiation sterilization for decellularized tracheal grafts
Uploaded: 2023-04-14T13:00:00
Description: Watch this Scientific Journal Video about Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts at JoVE.com

This paper was supported by the 2018 Spanish Society of Thoracic Surgery Grant to National Multicentric Study [Number 180101 awarded to Ne&#769;stor J.Marti&#769;nez-Herna&#769;ndez] and PI16-01315 [awarded to Manuel Mata-Roig] by the Instituto de Salud Carlos III. CIBERER is funded by the VI National R&#38;D&#38;I Plan 2018-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund.

The European directive 20170/63/EU for the care and use of laboratory animals was adhered to and the study protocol was approved by the Ethics Committee of the University of Valencia (Law 86/609/EEC and 214/1997 and Code 2018/VSC/PEA/0122 Type 2 of the Government of Valencia, Spain).
1. Tracheal decellularization
NOTE: The decellularization method has been reported elsewhere20.
<ol>
	<li>Euthanize donor male adult New Zeala...

<ol>
	<li>Ch'ng, 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=Reconstruction+of+the+(Crico)trachea+for+malignancy+in+the+virgin+and+irradiated+neck.">Reconstruction of the (Crico)trachea for malignancy in the virgin and irradiated neck.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 65 (12), 1645-1653 (2012).</li><li>Johnson, C. M., Guo, D. H., Ryals, S., Postma, G. N., Weinberger, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+feasibility+of+gamma+radiation+sterilization+for+decellularized+tracheal+grafts.">The feasibility of gamma radiation sterilization for decellularized tracheal grafts.</a> Laryngoscope. 127 (8), 258-264 (2017).</li><li>de Donato, 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=Prosthesis+infection:+prevention+and+treatment.">Prosthesis infection: prevention and treatment.</a> The Journal of Cardiovascular Surgery. 55 (6), 779-792 (2014).</li><li>Vangsness, C. T., Dellamaggiora, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+safety+sterilization+and+tissue+banking+issues+for+soft+tissue+allografts.">Current safety sterilization and tissue banking issues for soft tissue allografts.</a> Clinics in Sports Medicine. 28 (2), 183-189 (2009).</li><li>Den Hondt, M., Vanaudenaerde, B. M., Delaere, P., Vranckx, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty+years+of+experience+with+the+rabbit+model,+a+versatile+model+for+tracheal+transplantation+research.">Twenty years of experience with the rabbit model, a versatile model for tracheal transplantation research.</a> Plastic and Aesthetic Research. 3 (7), 223-230 (2016).</li><li>Hysi, I., 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=Successful+orthotopic+transplantation+of+short+tracheal+segments+without+immunosuppressive+therapy.">Successful orthotopic transplantation of short tracheal segments without immunosuppressive therapy.</a> European Journal of Cardiothoracic Surgery. 47 (2), 54-61 (2015).</li><li>Wurtz, 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=Tracheal+reconstruction+with+a+composite+graft:+Fascial+flap-wrapped+allogenic+aorta+with+external+cartilage-ring+support.">Tracheal reconstruction with a composite graft: Fascial flap-wrapped allogenic aorta with external cartilage-ring support.</a> Interactive Cardiovascular and Thoracic Surgery. 16 (1), 37-43 (2013).</li><li>White, A., Burns, D., Christensen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+terminal+sterilization+using+supercritical+carbon+dioxide.">Effective terminal sterilization using supercritical carbon dioxide.</a> Journal of Biotechnology. 123 (4), 504-515 (2006).</li><li>Qiu, Q. Q., 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=Inactivation+of+bacterial+spores+and+viruses+in+biological+material+using+supercritical+carbon+dioxide+with+sterilant.">Inactivation of bacterial spores and viruses in biological material using supercritical carbon dioxide with sterilant.</a> Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 91 (2), 572-578 (2009).</li><li>Lange, 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=Pilot+study+of+a+novel+vacuum-assisted+method+for+decellularization+of+tracheae+for+clinical+tissue+engineering+applications.">Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (3), 800-811 (2017).</li><li>Wedum, A. G., Hanel, E., Phillips, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+sterilization+in+microbiological+laboratories.">Ultraviolet sterilization in microbiological laboratories.</a> Public Health Reports. 71 (4), 331-336 (1956).</li><li>Hennessy, R. 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=Supercritical+carbon+dioxide-based+sterilization+of+decellularized+heart+valves.">Supercritical carbon dioxide-based sterilization of decellularized heart valves.</a> JACC. Basic to Translational Science. 2 (1), 71-84 (2017).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Balestrini, J. L., 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=Sterilization+of+lung+matrices+by+supercritical+carbon+dioxide.">Sterilization of lung matrices by supercritical carbon dioxide.</a> Tissue Engineering. Part C, Methods. 22 (3), 260-269 (2016).</li><li>AENOR. UNE-EN. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISO+11737-1:2006.+Esterilizaci&#243;n+de+productos+sanitarios.+M&#233;todos+biol&#243;gicos.+Parte+1:+Determinaci&#243;n+de+la+poblaci&#243;n+de+microorganismos+en+los+productos.">ISO 11737-1:2006. Esterilizaci&#243;n de productos sanitarios. M&#233;todos biol&#243;gicos. Parte 1: Determinaci&#243;n de la poblaci&#243;n de microorganismos en los productos.</a> AENOR. UNE-EN. , (2006).</li><li>Uriarte, J. 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=Mechanical+properties+of+acellular+mouse+lungs+after+sterilization+by+gamma+irradiation.">Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation.</a> Journal of the Mechanical Behavior of Biomedical Materials. 40, 168-177 (2014).</li><li>Sun, W. Q., Leung, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetric+study+of+extracellular+tissue+matrix+degradation+and+instability+after+gamma+irradiation.">Calorimetric study of extracellular tissue matrix degradation and instability after gamma irradiation.</a> Acta Biomaterialia. 4 (4), 817-826 (2008).</li><li>Nguyen, H., 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=Reducing+the+radiation+sterilization+dose+improves+mechanical+and+biological+quality+while+retaining+sterility+assurance+levels+of+bone+allografts.">Reducing the radiation sterilization dose improves mechanical and biological quality while retaining sterility assurance levels of bone allografts.</a> Bone. 57 (1), 194-200 (2013).</li><li>Helder, M. R. K., 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=Low-dose+gamma+irradiation+of+decellularized+heart+valves+results+in+tissue+injury+in+vitro+and+in+vivo.">Low-dose gamma irradiation of decellularized heart valves results in tissue injury in vitro and in vivo.</a> The Annals of Thoracic Surgery. 101 (2), 667-674 (2016).</li><li>Mart&#237;nez-Hern&#225;ndez, N. 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=Decellularized+tracheal+prelamination+implant:+A+proposed+bilateral+double+organ+technique.">Decellularized tracheal prelamination implant: A proposed bilateral double organ technique.</a> Artificial Organs. 45 (12), 1491-1500 (2021).</li><li>Feldman, A. T., Wolfe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+processing+and+hematoxylin+and+eosin+staining.">Tissue processing and hematoxylin and eosin staining.</a> Methods in Molecular Biology. 1180, 31-43 (2014).</li><li>L&#243;pez Caballero, J., Pe&#241;a, M., De Federico, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coloraciones+para+fibras+col&#225;genas+y+el&#225;sticas+del+tejido+conjuntivo.+Coloraciones+para+sustancia+amiloidea.">Coloraciones para fibras col&#225;genas y el&#225;sticas del tejido conjuntivo. Coloraciones para sustancia amiloidea.</a> Laboratorio de Anatom&#237;a Patologica. , 175-195 (1993).</li><li>Mart&#237;nez-Hern&#225;ndez, N. 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=A+standardised+approach+to+the+biomechanical+evaluation+of+tracheal+grafts.">A standardised approach to the biomechanical evaluation of tracheal grafts.</a> Biomolecules. 11 (10), 1461 (2021).</li><li>Kajbafzadeh, A. M., Javan-Farazmand, N., Monajemzadeh, M., Baghayee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+optimal+decellularization+and+sterilization+protocol+for+preparing+a+tissue+scaffold+of+a+human-sized+liver+tissue.">Determining the optimal decellularization and sterilization protocol for preparing a tissue scaffold of a human-sized liver tissue.</a> Tissue Engineering. Part C, Methods. 19 (8), 642-651 (2013).</li><li>Wehmeyer, J. L., Natesan, S., Christy, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+sterile+amniotic+membrane+tissue+graft+using+supercritical+carbon+dioxide.">Development of a sterile amniotic membrane tissue graft using supercritical carbon dioxide.</a> Tissue Engineering. Part C, Methods. 21 (7), 649-659 (2015).</li><li>Ross, E. 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=Mouse+stem+cells+seeded+into+decellularized+rat+kidney+scaffolds+endothelialize+and+remodel+basement+membranes.">Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.</a> Organogenesis. 8 (2), 49-55 (2012).</li></ol>

There are several sterilization strategies that exist. Supercritical CO2fully penetrates tissues, acidifying the medium and deconstructing the cellular phospholipid bilayer with simple elimination by means of depressurization of the implant8,14,25. Ultraviolet radiation has also been used, and its effectiveness in rodent trachea has been published, although there are only a few reports in the literature<sup class="xre...

Sterilization of an implant is a basic requisite for its viability; in fact, prostheses that have proven to be successful are those implanted in sterile areas (blood vessels, heart, bone, etc.)1. The trachea has two surfaces: a surface in contact with the external environment, which is therefore not sterile, and a surface toward the mediastinum, which is sterile. Therefore, from the moment the trachea is extracted, it is not a sterile organ. Despite the subsequent decellularization process being carried out in maximum sterile conditions, it is not a sterilization step2. The implantation of foreign material in itself entails a risk of infection due to the probacterial microenvironment it produces3and an up to 0.014% risk of disease transmission from the donor to the recipient, even if the material has been sterilized4. To ensure correct vascularization of the trachea, in almost all experimental transplant protocols, it first undergoes heterotopic implant5,6,7&#160;to a sterile area (muscle, fascia, omentum, subcutaneous, etc.); this is because implanting a non-sterile element in this medium would lead to infection of the area3.
There are a range of possible strategies to obtain a sterile implant. Using supercritical CO2has achieved terminal sterilization8,9. Other methods, such as ultraviolet radiation or treatment with substances such as peracetic acid, ethanol, oxygen peroxide, and electrolyzed water, have obtained different success rates in sterilization, almost always depending on their dosages, but they have been shown to affect the biomechanical characteristics of implants. Indeed, some substances, such as ethylene oxide, can substantially change the structure of the implanted matrix and can even cause undesirable immunogenic effects. For this reason, many of these strategies cannot be applied to biological models2,10,11,12,13.
The most widely studied and accepted sterilization strategy is that established by the ISO 11737-1:2006 standard for the sterilization of medical devices implanted in humans, with a gamma radiation dose of 25 kGy. However, this regulation focuses only on the sterilization of inert, non-biological elements14,15. Additionally, radiotherapy doses in the radical treatment of carcinoma are three orders of magnitude lower than those used to sterilize medical devices1. With this in mind, we can conclude that said dose would not only kill the microbiota but would also destroy and radically alter the biological structure of the implant. There is also the possibility that it would generate residual lipids upon degradation, which can potentially be cytotoxic and accelerate the enzymatic degradation of the scaffold13,14,15,16,17, even when using doses as low as 1.9 kGy and with damage directly proportional to the radiation dose received17.
Thus, the objective of this paper is to try to identify the radiation dose that allows for a sterile implant to be obtained with minimal harmful effects caused by irradiation2,18,19. The strategy we followed involved the irradiation of decellularized and irradiated tracheas at different escalated doses within a range of kilograys (0.5, 1, 2, 3 kGy, etc.), until achieving a negative culture. Additional tests were carried out for those doses that achieved negative cultures, in order to confirm sterilization. After determining the minimum dose to obtain sterilization, the structural and biomechanical impact of the irradiation on the trachea were checked. All the metrics were compared with the control native rabbit tracheas. The sterilization of the construct was then tested in vivo by implanting the tracheas into New Zealand white rabbits.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-0 nylon monofilament suture&#160;</td>
			<td>Monosoft. Covidien; Mansfield, MA, USA</td>
			<td>SN-5698G</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B 5%</td>
			<td>Gibco Thermo Fisher Scientific; Waltham, MA USA</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent, Santa Clara, CA, USA</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Buprex. Reckitt Benckiser Healthcare; Hull, Reino Unido</td>
			<td>N02AE01</td>
			<td></td>
		</tr>
		<tr>
			<td>Compression desktop UTM</td>
			<td>Microtest, Madrid, Spain</td>
			<td>EM1/10/FR</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostate</td>
			<td>Leyca CM3059, Leyca Biosystems, Wetzlar, Alemania</td>
			<td>CM3059</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamino-2-phenylindole)&#160;</td>
			<td>DAPI. Sigma-Aldrich, Missouri, USA&#160;</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich; MO, USA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Thermo Fisher Scientific; Waltham, MA, USA</td>
			<td>11965084</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA extraction kit</td>
			<td>DNeasy extraction kit Quiagen, Hilden, Germany</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacin, 2.5%</td>
			<td>Boehringer Ingelheim, Ingelheim am Rhein, Germany</td>
			<td>0035-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GE Healthcare Hyclone; Madrid, Spain</td>
			<td>SH20898.03IR</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leyca DM2500 (Leica, Wetzlar, Germany)</td>
			<td>DM2500??</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing Container&#160;</td>
			<td>Mr Frosty. Thermo Fisher; Madrid, Spain</td>
			<td>&#160;5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Isoflo; Proyma Ganadera; Ciudad Real, Spain&#160;</td>
			<td>8.43603E+12</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Imalgene. Merial; Toulouse, Francia</td>
			<td>BOE127823</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear accelerator&#160;</td>
			<td>&#34;True Beam&#34;. Varian, Palo Alto, California, USA</td>
			<td>H191001</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>Orbital Shaker PSU-10i. Biosan; Riga, Letonia</td>
			<td>BS-010144-AAN</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam 5 mg/ml</td>
			<td>Boehringer Ingelheim, Ingelheim am Rhein, Germany</td>
			<td>6283-MV</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT (Optimal Cutting Temperature Compound)</td>
			<td>Fischer Scientific, Madrid, Spain</td>
			<td>12678646</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 5%</td>
			<td>Gibco Thermo Fisher Scientific; Waltham, MA USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Dolethal. Vetoquinol; Madrid, Espa&#241;a</td>
			<td>3.60587E+12</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich; MO, USA</td>
			<td>P2272</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Propofol Lipuro. B. Braun Melsungen AG; Melsungen, Alemania</td>
			<td>G 151030</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Gibco Thermo Fisher Scientific; Waltham, Massachussetts, USA</td>
			<td>S3020</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC hollow tubes</td>
			<td>Cristallo Extra; FITT, Sandrigo, Italy</td>
			<td>hhdddyyZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC stent&#160;</td>
			<td>ArgyleTM Medtronic; Istanbul, Turkey</td>
			<td>019 5305 1</td>
			<td></td>
		</tr>
		<tr>
			<td>R software, Version 3.5.3 R Core</td>
			<td>R Foundation for Statistical Computing</td>
			<td>R 3.5.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich; MO, USA</td>
			<td>8,17,034</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Nanodrop, Life Technologies; Isogen Life Science. Utrech, Netherlands</td>
			<td>ND-ONEC-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet</td>
			<td>Microsoft Excel for Mac, Version 16.23, Redmond, WA, USA</td>
			<td>2864993241</td>
			<td></td>
		</tr>
		<tr>
			<td>Traction Universal Testing Machine&#160;</td>
			<td>Testing Machines, Veenendaal, Netherlands</td>
			<td>84-01</td>
			<td></td>
		</tr>
		<tr>
			<td>UTM Software</td>
			<td>TestWorks 4, MTS Systems Corporation, Eden Prairie, MN, USA&#160;</td>
			<td>100-093-627 F</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium&#160;</td>
			<td>Vector Labs, Burlingame; CA; USA</td>
			<td>H-1000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylacine</td>
			<td>Xilagesic. Calier; Barcelona, Espa&#241;a</td>
			<td>20102-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

low-dose gamma radiation sterilization for decellularized tracheal grafts

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation involves implanting an organ that was originally in contact with the environment, thus not being sterile from the outset. While the decellularization protocol (through detergent exposition [2% sodium dodecyl sulfate], continuous stirring, and osmotic shocks) is conducted in line with aseptic measures, it does not provide sterilization. Therefore, one of the main challenges is ensuring sterility prior to in vivo implantation. Although there are established gamma radiation sterilization protocols for inorganic materials, there are no such measures for organic materials. Additionally, the protocols in place for inorganic materials cannot be applied to organic materials, as the established radiation dose (25 kGy) would completely destroy the implant. This paper studies the effect of an escalated radiation dose in a decellularized rabbit trachea. We maintained the dose range (kGy) and tested escalated doses until finding the minimal dose at which sterilization is achieved. After determining the dose, we studied effects of it on the organ, both histologically and biomechanically. We determined that while 0.5 kGy did not achieve sterility, doses of both 1 kGy and 2 kGy did, with 1 kGy, therefore, being the minimal dose necessary to achieve sterilization. Microscopic studies showed no relevant changes compared to non-sterilized organs. Axial biomechanical characteristics were not altered at all, and only a slight reduction in the force per unit of length that the organ can radially tolerate was observed. We can therefore conclude that 1 kGy achieves complete sterilization of decellularized rabbit trachea with a minimal, if any, effects on the organ.

Decellularization 
DAPI staining shows the absence of DNA, and no DNA values higher than 50 ng were detected in any of the tracheae any by electrophoresis, with all fragments being smaller than 200 bp20.
Microbial culture 
Two of the eight pieces subjected to 0.5 kGy showed color change in less than 1 week. None of the pieces irradiated at 1 kGy and 2 kGy showed any color change (Figure 1).
<p...

Watch this Scientific Journal Video about Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts at JoVE.com

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

Obtaining sterilization is essential for tracheal tissue transplant. Herein, we present a sterilization protocol using low-dose gamma irradiation that is fully tolerated by organs.

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation ...

This protocol provides a complete sterilization process for any non-sterile organ without affecting its characteristics. The main objective of this technique includes the possibility of sterilizing an organ without affecting its properties. Although this technique has focused on tracheas, it has demonstrated that lower gamma radiation doses can effectively sterilize biological materials.An individual may face the problem by defining the lowest effective dose for the different type of organs. Then, it's recommended to begin with a lower dosage and increase them until the sterilization is achieved. Begin by sectioning the trachea dissected from euthanized New Zealand white rabbits, or Oryctolagus cuniculus, into two-centimeter pieces.With scissors, remove the surrounding connective tissue and inner mucosal layer. Submerge the specimens in 12 milliliters of PBS solution containing SDS, 5%penicillin streptomycin, and 5%amphotericin. Subject the tracheas to constant stirring with a magnetic stirrer at 400 RPM for five weeks at room temperature.Weekly, give two hours of osmotic shock by immersing the tracheas in distilled water before changing the medium. After five weeks, cryogenize the trachea specimens using a 12-milliliter mixture of 80%FBS and 20%DMSO in a freezing container at minus 80 degrees Celsius. After 13 to 15 days, thaw the tracheas in a water bath at 37 degrees Celsius.Then, wash them with PBS. To irradiate the specimen, place four tracheal pieces in 20 milliliters of PBS in methacrylate T25 culture flasks, while preventing bubble formation. Conduct the irradiation using a linear accelerator with photons of a nominal energy of 10 megavolts flattening filter-free beams.Apply a dose rate of 2, 400 monitor units per minute and set other parameters as described in the manuscript. Once done, culture the specimen in 30 milliliters of DMEM supplemented with 10%inactivated FBS without antibiotics or antifungals. Incubate the culture in a standard tissue incubator at 37 degrees Celsius and 5%carbon dioxide for two weeks and inspect it every 24 hours for contamination parameters like medium pH, color, and turbidity changes.Conduct tensile tests on a traction desktop universal testing machine, or UTM displacement control, equipped with force and position sensors and a computer with specifically-designed software. Record data every 0.4 seconds and export it to a spreadsheet. Construct tensile jaws adapted to the mean caliber of the rabbit tracheas from pure monolayer non-toxic crystal polyvinyl chloride, or PVC hollow tubes.Section the conducts into three-centimeter-long segments. To perform the termino-terminal suture without bias, drill 12 preformed holes two millimeters from the edge of the jaws, separated by 2.5 millimeters. Next, attach the PVC glass tubes to the rabbit trachea by termino-terminal anastomosis with a continuous 6-0 nylon monofilament suture through alternate-performed five-millimeter holes present two millimeters away from the edge of the trachea.Stretch all the pieces at a displacement rate of 5.0 millimeters per minute. Then, record the variables, such as maximum stress and strain, the energy stored per unit of trachea volume, and Young's modulus. Perform radial compression tests on a compression desktop UTM equipped with a 15-newtons load cell to obtain force data, position, and time.Record data and export to spreadsheets at 0.5 second intervals. Place the tracheas with the membranous area resting on the lower plate before the gradual rise of the plate toward the top plate at a constant speed of five millimeters per minute. Calculate every variable per unit of length of the sample, stiffness, and the energy per unit of the surface area needed to occlude the trachea completely.Place a sterile intraluminal PVC stent of size 14, ensuring a three-to four-millimeter margin at each end. Fix the stent with a single 6-0 nylon monofilament stitch through the intercartilaginous space of the first cartilage. Under aseptic conditions and with sterile material, make a longitudinal three-centimeter central thoracic incision and harvest bilateral pedicled flaps composed of pectoral fascia and a muscular component.Wrap the tracheas with the flap in four rabbits, one trachea on each hemathorax. Once the surgery is completed, reverse the anesthesia by interrupting isoflurane administration. Out of the eight pieces that were exposed to 0.5 kilogray of radiation, two pieces exhibited a change in media color within one week.However, none of the pieces that were irradiated at one or two kilogray showed any change in media color. Compared to the control, no collagen or elastic fiber distribution pattern changes were detected in the analyzed specimens irradiated at different radiation doses. The data obtained in the tensile test on irradiated tracheas is shown here.Stress strain curves for decellularized and irradiated tracheas revealed no changes compared to non-sterilized tracheas. The compression tests performed on the native tracheas or controls and the decellularized, cryopreserved, and irradiated tracheas are shown. The corresponding graphs showed that irradiation did not change the percentage of occlusion compared to native trachea.Gamma irradiation caused a clinically-minimal but statistically-significant decrease in radio-biomechanical characteristics and the variable force per unit of length. The histological examination showed the highly-organized connective tissue in the form of the perichondrium of the native trachea. The cartilage was intact and showed no signs of necrosis.When implanting radiological element in a living being, the sterility of the implant and the sterile process are paramount to avoid infection and tissue rejection. There's a broad range of organs to be implanted after tissue generating, and they should be sterile. Following this process can achieve a complete sterilization without affecting the main characteristics of the organ.

low-dose-gamma-radiation-sterilization-for-decellularized-tracheal

Research

JoVE Journal

Bioengineering

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 ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

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 ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Soybean Seed Sterilization

To begin, place 16 to 20 pristine round Williams 82 soybean seeds in a 50 milliliter centrifuge tube inside a biosafety cabinet. Add 30 milliliters of 70%isopropyl alcohol to the tube and shake for 30 seconds. Decant the alcohol.Next, gently shake the seeds with 30 milliliters of 10%bleach for 10 seconds, and allow the seeds to sit in the solution for 5 minutes at room temperature. After 5 minutes, drain the bleach then rinse the seeds three times with 30 milliliters of sterile ultrapure water for 1 minute per rinse and discard the water between each rinse. Place the sterilized seeds on filter paper saturated with 5 milliliters of germination and co-cultivation medium in a sterile petri dish.Place the plates in the dark at room temperature for 3 days before transferring them at 22 degrees Celsius under 16 hour cool white T5 fluorescent lights for 4 days to allow the seeds to germinate.

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation. Ascertaining novel bioengineering approaches for the replacement of diseased lungs is imperative for improving patient survival and avoiding complications associated with current transplantation methodologies. An alternative approach involves the use of decellularized whole lungs lacking cellular constituents that are typically the cause of acute and chronic rejection. Since the lung is such a complex organ, it is of interest to examine the extracellular matrix components of specific regions, including the vasculature, airways, and alveolar tissue. The purpose of this approach is to establish simple and reproducible methods by which researchers may dissect and isolate region-specific tissue from fully decellularized lungs. The current protocol has been devised for pig and human lungs, but may be applied to other species as well. For this protocol, four regions of the tissue were specified: airway, vasculature, alveoli, and bulk lung tissue. This procedure allows for the procurement of samples of tissue that more accurately represent the contents of the decellularized lung tissue as opposed to traditional bulk analysis methods.

Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue  

Presented here is a protocol for the isolation of regional decellularized lung tissue. This protocol provides a powerful tool for studying complexities in the extracellular matrix and cell-matrix interactions.

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...