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>
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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. 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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. 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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.

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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).
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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.

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