Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

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.