Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 implant^5,6,7 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 sterilization^8,9. 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 models^{2,10,11,12,13}.

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 elements^14,15. 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 scaffold^{13,14,15,16,17}, 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 irradiation^2,18,19. 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.

Protokół

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.

1. Euthanize donor male adult New Zealand white rabbits (Oryctolagus cuniculus) weighing 3.5-4.1 kg with 133 mg/kg pentobarbital sodium, using a 200 mg/mL injection through the marginal ear vein.
2. While ensuring aseptic conditions, perform a central longitudinal cervicotomy, dissect the cervical muscles, and approach the trachea. Dissect the organ circumferentially and longitudinally. Finally, transect under the first ring and just above the carina.
3. With a scalpel, section the tracheas into 2 cm pieces. With scissors, remove the surrounding connective tissue and inner mucosa layer⁶.
4. Submerge the specimens in 12 mL of phosphate buffered saline (PBS) solution containing 2% sodium dodecyl sulfate (SDS), 5% penicillin-streptomycin, and 5% amphotericin B.
5. Subject the tracheas to constant stirring with a magnetic stirrer at 400 rpm for 5 weeks at room temperature. Replace the decellularization solution weekly after a 2 h osmotic shock, by means of immersion of the trachea in distilled water.
6. Cryogenize the specimens using a 12 mL mixture of 80% fetal bovine serum (FBS) and 20% dimethyl sulfoxide (DMSO) in a freezing container at -80 °C.
7. When the tracheas are going to be used (after 13-15 days), thaw them in a water bath at 37 °C and wash them by submerging them in PBS after thawing is complete.

2. Sterilization

1. Irradiation
   1. Place batches of four tracheal pieces measuring 2 cm each in a 20 mL of methacrylate in T25 culture flask filled with PBS until a total volume of 30 mL is reached. Take care to prevent bubbles from forming, which could cause energy diffusion in the air-liquid interface.
   2. Conduct irradiation using a linear accelerator, with photons of a nominal energy of 10 MV flattening filter free beams. Apply a dose rate of 2,400 monitor units per minute at the isocenter, placing the tracheas at a source-surface distance of 100 cm to be irradiated, with a depth of field of 2.5 cm for a radiation field of 10 cm x 10 cm-so as to cover the entire container-corresponding to a dose of 24 Gy/min.
   3. Escalate the doses with each four piece batch; four pieces will be subjected to 0.5 kGy, four to 1 kGy, four to 2 kG, etc., until sterilization is reached.
2. Culture
   1. Introduce the pieces into 30 mL of Dulbecco's modified Eagle's medium (DMEM) with inactivated 10% FBS without antibiotics or antifungals.
   2. Culture them in a standard tissue incubator at 37 °C and 5% CO₂ for 2 weeks and inspect them every 24 h.
      NOTE: The contamination parameters are changes in the pH of the culture medium and, accordingly, changes in the color and turbidity of the medium. The tracheas were harvested from germ-free lagomorphs, which were not ill and, thus, expected to be devoid of anaerobic bacteria in their tracheas.

3. Histological analysis

NOTE: Stain the pieces using hematoxylin and eosin²¹, Masson's trichrome, and orcein²².

1. DAPI staining
   1. Determine the tissue viability using DAPI (4′,6-diamidino-2-phenylindole). This blue-florescent stain binds strongly to adenine- and thymine-rich regions in DNA sequences, and therefore allows for DNA to be viewed via fluorescence microscopy.
   2. Embed the tissue samples in optimal cutting temperature (OCT) compound.
   3. Cut the samples using a cryostat.
   4. Wash the sample to be dyed three times in distilled water to remove the OCT. Place in mounting medium that includes a 30 nM solution of DAPI.
   5. Visualize fluorescence using fluorescence microscopy.
2. DNA content analysis
   1. Cut segments of trachea measuring approximately 3 mm long using a scalpel.
   2. Incubate for 2 h in proteinase K (Table of Materials).
   3. Extract the DNA with a DNA extraction kit, following the manufacturer's instructions.
   4. By means of spectrophotometry, determine the concentration of DNA by measuring the absorbance at 260/280 using a spectrophotometer.
   5. Measure the size of the extracted DNA samples using capillary chromatography with a bioanalyzer.

4. Biomechanical study

NOTE: Tracheal resistance to longitudinal and transverse forces is measured through axial tensile and radial compression tests²³.

1. Tracheal measurement
   1. Measure the tracheal length, wall thickness, and external diameter using a Vernier caliper.
   2. Calculate the average values from three random measurements of each of the variables.
   3. In the radial compression tests, calculate the anteroposterior diameter by detecting the point at which the plate comes into contact with the specimen.
   4. Perform all tests at room temperature.
2. Tensile tests
   1. Conduct tensile tests on a traction desktop universal testing machine (UTM) displacement control, equipped with a 100 N load (0.1 N force resolution, 0.001 mm of position, and 0.1 s). The testing machine is equipped with force and position sensors, and is connected to a computer with software specifically designed by the manufacturer²³.
   2. Record data every 0.4 s and export to a spreadsheet.
   3. Construct tensile jaws adapted to the mean caliber of the rabbit tracheas from pure monolayer, non-toxic crystal polyvinyl chloride (PVC) hollow tubes with an external diameter of 1 cm and a wall thickness of 1.5 mm.
   4. Section the conducts into 3 cm long segments.
   5. Drill 12 preformed holes for the termino-terminal suture, 2 mm from the edge of the jaws and separated by a distance of 2.5 mm, to prevent bias due to the sutures.
   6. Attach the PVC glass tubes to the rabbit trachea by termino-terminal anastomosis with a continuous suture through alternate preformed holes (every 5 mm), 2 mm away from the edge of the trachea and with a 6-0 nylon monofilament suture.
   7. Stretch all the pieces at a displacement rate of 5.0 mm/min.
   8. Record the variables maximum stress (σ_max, in N/mm²) and strain (ε_max, with no units), together with the energy stored per unit of trachea volume (W/Vol, in mJ/mm), and Young's modulus (E, in MPa).
3. Radial compression tests
   1. Perform radial compression tests on a compression desktop UTM, equipped with a 15 N load cell (force resolution 0.001 N, position 0.001 mm, and time 0.1 s) to obtain force data (N), position (mm), and time (s). Record data and export to spreadsheet at 0.5 s intervals.
   2. Place the tracheas with the membranous area resting on the lower plate. The plate gradually rises up toward the top plate at a constant speed of 5 mm/min.
   3. Calculate every unit per unit of length of the sample (f in N/mm), stiffness (R in Mpa·mm), and the energy per unit of surface area (W / S in mJ/mm²) needed to completely occlude the trachea.

5. Surgical technique

NOTE: The surgical technique has been widely reported elsewhere²⁰.

1. Place a sterile intraluminal PVC stent, size 14 Fr (which allows it to slide freely without compressing the walls), with a 3-4 mm margin at each end.
2. Fix the stent with a single 6-0 nylon monofilament stitch through the intercartilaginous space of the first cartilage.
3. Proceed to anesthetize the rabbits.
   1. Pre-medicate the subjects (3.65-4.05 kg male New Zealand white rabbits) with intramuscular analgesics (35 mg/kg ketamine) with a sedative, muscle relaxant, and analgesic (2.5 mg/kg xylazine).
   2. Shave the incision zone out of the operating zone and clean the surgical area to remove the hair.
   3. Administer analgesics plus antibiotic prophylaxis: 0.05 mg/kg intramuscular buprenorphine and 10 mg/kg enrofloxacin.
   4. Put a venous catheter in the marginal ear vein of each rabbit.
   5. Induct anesthesia with an intravenous 10 mg/kg bolus of propofol.
   6. Monitor the animal's vital signs using a three-lead electrocardiogram, pulse oximetry, and noninvasive pressure measurement. Every 30 min, apply physiological serum to the eyes to prevent dryness while under anesthesia.
   7. Verify the anesthetic plane using the toe pinch method.
   8. Maintain anesthesia with inhaled isoflurane at 1.5%-2% of the minimum alveolar concentration without losing spontaneous ventilation and provide thermal support to the rabbit with a heating pad.
4. Disinfect the incision zone several times in a circular motion with an iodine-based scrub. Under aseptic conditions at all times and with sterile material, make a longitudinal 3 cm central thoracic incision, and harvest bilateral pedicled flaps composed of pectoral fascia and a muscular component.
5. Wrap the tracheas with the flap in four rabbits, one on each hemithorax (thus, a total of eight tracheas).
6. When the surgery is completed, reverse the anesthesia by interrupting isoflurane administration.
7. Postsurgical period
   1. Keep the animals in the operating room until they have completely recovered from anesthesia. When they have completely recovered, return them to their environment with other rabbits.
   2. Treat the rabbits with antibiotics (0.5 mL/kg of 2.5% enrofloxacin) and analgesics (5 mg/mL meloxicam; 0.05 mL/kg metacam) every 24 h for 5 days.
   3. Leave the implants in situ for the desired time.
   4. Prior to euthanasia, pre-medicate the rabbits with intramuscular analgesics (35 mg/kg ketamine) and a sedative, muscle relaxant, and analgesic (2.5 mg/kg xylazine). Then euthanize the rabbits with 133 mg/kg pentobarbital sodium using a 200 mg/mL injection through the marginal ear vein and harvest the tracheas.
   5. Perform biomechanical and histological tests on the tracheas.

6. Statistical analysis

1. Adjust all the models by the Bayesian method on R software, Version 3.5.3 R Core (R Foundation for Statistical Computing. 2019).
2. Analyze the study variables, except f and R, using multiple linear regression models.
3. For the f and R variables, apply mixed linear regression models. In these models, in addition to the variables of interest related to the treatment and condition of each trachea, introduce the percentage of occlusion as a monotonic effect and an independent term per trachea as a random factor.

Wyniki

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).

Dyskusje

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 implant^8,14,25. 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

Materiały

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