Irradiation Types

The energy of all ionizing radiation is expressed as electron volts (eV) and is defined as energy gained by an electron moving through a potential difference of 1 volt. Need to move up? The energy absorbed by a substance, such as a soft-tissue graft is measured in gray (Gy): 1 Gy = 1 J/kg. In older publications, a previously used unit was rad: 1 rad = 10 ⁻² J/kg. Thus 1 Gy = 100 rad or, for example, 25 kGy = 2.5 Mrad.

Ionizing radiation can be initiated by various sources. In today’s practice, the only sources used are ₆₀ Co (photon energy levels 1.17 MeV and 1.33 MeV) which creates gamma rays and accelerated electrons, which create electron beams (8–10 MeV). Commercial gamma ray irradiators are loaded with ₆₀ Co of a total activity of 0.3–3.0 MCi, while commercial electron beam irradiators are equipped with one or two accelerators that generate high power (10–100 kW) beams of 8–10 MeV electrons.

Gamma irradiation is still today’s most commonly used technique for soft-tissue sterilization, even though electron beam irradiation is currently becoming more popular. Specific differences between both radiation types exist. Gamma rays are high-energy photons, which have no mass and charge, and can therefore penetrate materials and tissue over a large distance.

Electron beam emitted electrons have mass and charge, which limits their penetrability depending on their energy, atomic number, and density of the processed tissue. Soft-tissue grafts of a thickness greater than 5 cm cannot be fully penetrated. The advantages of electron beam irradiation are its high effectiveness, lack of heat induction during the sterilization procedure, and absence of pressure differences and diffusion barriers typically associated with chemical sterilization methods. It allows for a significantly improved control of the overall applied irradiation dose. It shows much less dose variation per area and time during the sterilization procedure and a substantially reduced processing time (seconds for electron beam versus several hours for gamma irradiation).

Tissue Sterility and Legal Regulations

National legal authorities regulate the use of allografts and the steps necessary for tissue harvest, processing, storage, and transplantation. In the United States, for example, the Food and Drug Administration and its legal body, the Center for Biologics Evaluation and Research, established guidelines in 2005 for tissue banks that manufacture human tissue. Self-regulatory bodies, such as the American Association of Tissue Banks, accredit tissue banks that adhere to the regularly updated guidelines for tissue processing and standards of good clinical practice. Equivalent institutions exist in Europe, Asia, and Australia. Important differences exist with respect to tissue handling and processing, with some countries requiring tissue sterilization (e.g., Germany), while others do not (e.g., United States). The details of national legal requirements can be obtained from each country’s national regulatory institutions. All regulatory authorities are in agreement regarding the definition of tissue sterility. The concept of the sterility assurance level (SAL) is derived from kinetic studies on microbial inactivation—that is, the probability of viable microorganisms (active pathogens) to be present after sterilization. The SAL for human tissues is accepted at 10 ⁻⁶ . That means that the probability of a microorganism remaining on a sterilized tissue graft is, at most, 1 in 1 million. A SAL of 10 ⁻⁶ determines the radiation dosages to meet this standard for respective pathogens and types of tissue.

Inactivation Mechanism of Irradiation

Ionizing radiation is targeting the nucleic acids (DNA and RNA) of microorganisms. These are either directly damaged by the ionizing radiation or indirectly through radiolysis of water and the production of short-lived hydroxyl radicals. In the presence of oxygen, radicals form peroxide radicals and peroxides, which cause scissoring of DNA cross-links and damage to the DNA bases and sugars. This inhibits DNA synthesis, eventually. Depending on the dose of radiation, cells can repair the resulting damages. Therefore to obtain true tissue sterility, different pathogens require different irradiation doses, which, in turn, affect tissue properties. Common bacterial pathogens can be safely eliminated at doses of 15–20 kGy. Viruses, such as HIV, require 30 kGy, and small viral pathogens, such as parvovirus B19, necessitate 34 kGy, while prions might not even be eliminated at significantly higher doses.

Dose-Dependent Effects of Irradiation on Tendons and Ligaments

While free radical formation is important for pathogen inactivation, it also influences the integrity of allogenic tissue. It has been shown that increased irradiation doses have a detrimental effect on the biomechanical properties of soft-tissue grafts. The underlying effects of this observation are not fully understood. Several studies found that radical formation led to changes in collagen ultrastructure, cross-link density, and collagen and water content. The impact of gamma irradiation on the biomechanical properties of soft-tissue grafts, used in anterior cruciate ligament (ACL) reconstructions, has been analyzed in numerous studies. It was shown that dosages of gamma rays less than 20 kGy did not significantly alter biomechanical properties compared with nonirradiated soft-tissue grafts, such as bone–patellar tendon–bone (BPTB), anterior tibialis, semitendinosus tendons, or fascia lata grafts. However, gamma irradiation greater than 20 kGy resulted in significantly reduced structural, material, and viscoelastic properties of soft-tissue grafts.

Electron beam irradiation also showed a dose-dependent effect, even though less pronounced than gamma irradiation. Hoburg et al. examined paired BPTB grafts with one graft sterilized using electron beam irradiation and the addition of carbon dioxide as a radical scavenger, while the contralateral graft was left nonsterilized. The authors found no significant reduction in structural and viscoelastic properties of BPTB grafts at 15 and 25 kGy, compared with nonirradiated grafts. Only at 34 kGy was a significant reduced failure load observed. On the contrary, Seto et al. found similarly decreased structural properties for gamma and e-beam sterilized soft-tissue grafts compared with nonsterilized grafts at 25 and 50 kGy. However, no radical scavengers were added in their study for either irradiation technique. Viscoelastic properties were also not affected, similar to the findings by Hoburg et al. pointing out a possible advantage of electron beam versus gamma irradiation.

In summary, basic science studies showed a clear dose-dependent effect of ionizing irradiation on the biomechanical properties of soft-tissue grafts independently from the type of irradiation. The currently accepted dose threshold to prevent significant mechanical graft impairment is between 15 and 20 kGy.

Optimization Methods for Irradiation Techniques

Several methods exist that are aimed to reduce or even eliminate the detrimental effects of high-dose irradiation on the biomechanical and biological properties of soft-tissue grafts. It was shown that biomechanical properties of deep-frozen grafts were better preserved during high-dose (35 kGy) gamma irradiation compared with lyophilized or glycerolized grafts. Seto et al. looked at the effect of radical scavengers, such as mannitol, ascorbate, and riboflavin, to improve the biomechanical properties and collagenase resistance of rabbit Achilles tendons during gamma and electron beam irradiation. They found a protective effect at 25 kGy; however, there were only small effects at 50 kGy, with no differences between both irradiation types. Better radioprotection was achieved by the added cross-linkers, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and glucose, especially at higher irradiation doses of 50 kGy. The combination of radical scavengers and cross-linkers showed further radioprotection at high doses of 50 kGy of gamma and electron beam irradiation, with no differences found for tensile strength, elastic modulus, and failure strain, compared with untreated controls. Based on their positive findings, Seto et al. used an in vivo model to assess the effect of radioprotectants on graft healing. They reconstructed the ACL in sheep with allografts that were either radioprotected, as in their previous studies, or untreated during gamma irradiation of 50 kGy. Nonirradiated grafts functioned as controls. At 12 weeks, radioprotected irradiated grafts displayed subfailure mechanical properties and anterior tibial translation comparable to nonirradiated allografts. At 24 weeks, however, structural properties of radioprotected grafts were significantly lower than in nonirradiated controls, and histological analysis showed significantly delayed healing, especially at the tendon-bone interface. Therefore the authors concluded that further research into the effect of radioprotectants is needed before recommending their general use with human tissue.

Genipin is another cross-linker that has been examined as a radioprotectant. Genipin has low tissue toxicity and was shown to improve mechanical properties of pericardium, cornea, intervertebral discs, and cartilage in a time- and concentration-dependent fashion. Genipin is believed to introduce intermicrofibrillar cross-links between adjacent collagen microfibrils, thus enhancing the mechanical strength of such treated tissue. Ng et al. compared bovine and human patellar tendon strips (aged 47–56 years) treated with genipin and without at 50 kGy of gamma irradiation. Genipin treatment before irradiation improved tensile modulus of bovine tendons by 2.4-fold and showed no differences after 50-kGy irradiation compared with controls. The tensile modulus of genipin-treated human patellar tendons improved by 1.3-fold before irradiation, but it showed a 50% reduction following irradiation. No conclusive explanations were given for their findings, but the authors suggested that the higher age of human tissue donors might have played a role in the negative findings for human patellar tendons. These studies underscore the potential of radioprotective additives; however, further research is necessary to not only examine the time zero effects in vitro, but also the implications for the biological healing after ACL reconstruction and to separate possible differences between animal and human tissue.

Another approach to minimize the effect of high-dose irradiation is the fractionation of the overall dose into multiple smaller dosages. Fractionation of electron beam irradiation is a proven concept in tumor radiation and limits the exposure to tumor-free tissue. For damage reduction of the irradiated tissue, fractionation of the overall irradiation dosage has become a common procedure in radiotherapy. Further more, it has been shown that fractionation did not affect the efficiency of malignant cell elimination compared with single-dose therapy. Rationale for fractionation is a possible reduction of free radical formation, which would protect the tissue from biomechanical impairment. Hoburg et al. compared the structural and viscoelastic properties of human BPTB grafts that were irradiated with electrons at 34 kGy either in a single dose or fractionated in 10 doses of 3.4 kGy at time zero. These grafts were compared with 34 kGy gamma irradiated grafts and nonirradiated controls. The authors found no differences between fractionated e-beam irradiated and nonirradiated grafts, while single-dose irradiated grafts showed significantly lower biomechanical properties. Schmidt et al. looked at the effect of fractionated high-dose electron irradiation on ACL graft healing in a sheep model. The authors found significantly lower structural properties and increased anterior-posterior laxity at 6 and 12 weeks of healing for fractionated high-dose irradiated grafts compared with nonirradiated controls, thus not recommending fractionated high-dose irradiation for ACL graft sterilization. The findings of this study further underscore the importance of gaining a better understanding of the changes high-dose irradiation delivers to the composition and ultrastructure of the sterilized graft. It not only alters its biomechanical properties but also its behavior during the biological processes of graft healing and bony incorporation.