Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics

Gamma irradiation from Cobalt 60 sources has been used to terminally sterilize bone allografts for many years. Gamma radiation adversely affects the mechanical and biological properties of bone allografts by degrading the collagen in bone matrix. Specifically, gamma rays split polypeptide chains. In wet specimens irradiation causes release of free radicals via radiolysis of water molecules that induces cross-linking reactions in collagen molecules. These effects are dose dependent and give rise to a dose-dependent decrease in mechanical properties of allograft bone when gamma dose is increased above 25 kGy for cortical bone or 60 kGy for cancellous bone. But at doses between 0 and 25 kGy (standard dose), a clear relationship between gamma dose and mechanical properties has yet to be established. In addition, the effects of gamma radiation on graft remodelling have not been intensively investigated. There is evidence that the activity of osteoclasts is reduced when they are cultured onto irradiated bone slices, that peroxidation of marrow fat increases apoptosis of osteoblasts; and that bacterial products remain after irradiation and induce inflammatory bone resorption following macrophage activation. These effects need considerably more investigation to establish their relevance to clinical outcomes. International consensus on an optimum dose of radiation has not been achieved due to a wide range of confounding variables and individual decisions by tissue banks. This has resulted in the application of doses ranging from 15 to 35 kGy. Here, we provide a critical review on the effects of gamma irradiation on the mechanical and biological properties of allograft bone.     

Sterilization of Allograft Bone: is 25 kGy the Gold Standard for Gamma Irradiation?

For several decades, a dose of 25 kGy of gamma irradiation has been recommended for terminal sterilization of medical products, including bone allografts. Practically, the application of a given gamma dose varies from tissue bank to tissue bank. While many banks use 25 kGy, some have adopted a higher dose, while some choose lower doses, and others do not use irradiation for terminal sterilization. A revolution in quality control in the tissue banking industry has occurred in line with development of quality assurance standards. These have resulted in significant reductions in the risk of contamination by microorganisms of final graft products. In light of these developments, there is sufficient rationale to re-establish a new standard dose, sufficient enough to sterilize allograft bone, while minimizing the adverse effects of gamma radiation on tissue properties. Using valid modifications, several authors have applied ISO standards to establish a radiation dose for bone allografts that is specific to systems employed in bone banking. These standards, and their verification, suggest that the actual dose could be significantly reduced from 25 kGy, while maintaining a valid sterility assurance level (SAL) of 10⁻⁶. The current paper reviews the methods that have been used to develop radiation doses for terminal sterilization of medical products, and the current trend for selection of a specific dose for tissue banks.     

X-ray sterilization of biopharmaceutical manufacturing equipment—Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of ⁶⁰Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables—one concern connected with the use of single-use process equipment—the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.     

A murine model for bone loss from therapeutic and space-relevant sources of radiation.

Cancer patients receiving radiation therapy are exposed to photon (gamma/X-ray), electron, and less commonly proton radiation. Similarly, astronauts on exploratory missions will be exposed to extended periods of lower-dose radiation from multiple sources and of multiple types, including heavy ions. Therapeutic doses of radiation have been shown to have deleterious consequences on bone health, occasionally causing osteoradionecrosis and spontaneous fractures. However, no animal model exists to study the cause of radiation-induced osteoporosis. Additionally, the effect of lower doses of ionizing radiation, including heavy ions, on general bone quality has not been investigated. This study presents data developing a murine model for radiation-induced bone loss. Female C57BL/6 mice were exposed to gamma, proton, carbon, or iron radiation at 2-Gray doses, representing both a clinical treatment fraction and spaceflight exposure for an exploratory mission. Mice were euthanized 110 days after irradiation. The proximal tibiae and femur diaphyses were analyzed using microcomputed tomography. Results demonstrate profound changes in trabecular architecture. Significant losses in trabecular bone volume fraction were observed for all radiation species: gamma, (-29%), proton (-35%), carbon (-39%), and iron (-34%). Trabecular connectivity density, thickness, spacing, and number were also affected. These data have clear implications for clinical radiotherapy in that bone loss in an animal model has been demonstrated at low doses. Additionally, these data suggest that space radiation has the potential to exacerbate the bone loss caused by microgravity, although lower doses and dose rates need to be studied.     

Comparative sequence analysis of the eastern equine encephalitis virus pathogenic strains FL91-4679 and GA97 to other North American strains.

Eastern equine encephalitis (EEE) virus is a significant public health concern due to the high mortality rates observed in infected humans, equines and game birds. The EEE genomic sequences available prior to this report are based on laboratory strains with unknown passage histories that may contain an array of cell culture adaptations. Here we report the complete genomic sequences of two recently isolated EEE pathogenic strains with low passage histories. FL91-4697 was isolated in Florida from Aedes albopictus mosquitoes and GA97 was derived from brain tissue of a human fatality that occurred in 1997. Sequence alignment of these new genomes with the documented EEE's permitted us to generate a North American consensus sequence and identify regions of significant diversity. Sequence analysis of the FL91-4679 genome was essential to the production of an EEE infectious clone that is being used to create live attenuated vaccine candidates.     

Alterations in damage processes in dense cancellous bone following gamma-radiation sterilization

Structurally intact cancellous bone allograft is an attractive tissue form because its high porosity can provide space for delivery of osteogenic factors and also allows for more rapid and complete in-growth of host tissues. Gamma radiation sterilization is commonly used in cancellous bone allograft to prevent disease transmission. Commonly used doses of gamma radiation sterilization (25–35 kGy) have been shown to modify cortical bone post-yield properties and crack propagation but have not been associated with changes in cancellous bone material properties. The purpose of this study was to determine the effects of irradiation on the elastic and yield properties and microscopic tissue damage processes in dense cancellous bone. Cancellous bone specimens (13 control, 14 irradiated to 30 kGy) from bovine proximal tibiae were tested in compression to 1.3% apparent strain and examined for microscopic tissue damage. The yield strain in irradiated specimens (0.93±0.11%, mean±SD) did not differ from that in control specimens (0.90±0.11%, p=0.44). No differences in elastic modulus were observed between groups after accounting for differences in bone volume fraction. However, irradiated specimens showed greater residual strain (p=0.01), increased number of microfractures (p=0.02), and reduced amounts of cross-hatching type damage (p<0.01). Although gamma radiation sterilization at commonly used dosing (30 kGy) does not modify elastic or yield properties of dense cancellous bone, it does cause modifications in damage processes, resulting in increased permanent deformation following isolated overloading.     

Effect of gamma sterilization on microhardness of the cortical bone tissue of bovine femur in presence of N-Acetyl-L-Cysteine free radical scavenger

Gamma sterilization is usually used to minimize the risk of infection transmission through bone allografts. However, it is believed that gamma irradiation affects the mechanical properties of allografts and free radical scavengers can be used to alleviate the radiation-induced degradation of these properties. The aim of this study was to investigate the radioprotective effects of N-Acetyl-L-Cysteine (NAC) free radical scavenger on the material properties of sterilized bovine cortical bone at microstructure level. Forty-two cortical tissue specimens were excised from three bovine femurs and irradiated to 35 and 70 kGy gamma rays in the presence of 5, 50, and 100 mM concentrations of NAC. The localized variations in microhardness were evaluated via indentation in the radial and longitudinal directions to examine different regions of the microstructures of the specimens, including the osteonal and interstitial tissues. A significant increase was observed in the hardness of osteonal, interstitial, and longitudinal combined microstructures exposed to 35 and 70 kGy radiations (P < 0.05), whereas a relative reduction of the hardness was observed in the radial direction. Furthermore, it was found that the application of 50 and 100 mM NAC during gamma irradiation significantly subsided the hardening in longitudinal combined microstructure. Moreover, the reduction of hardness in radial direction was suppressed in the presence of 100 mM of NAC. In conclusion, the results indicated that NAC free radical scavenger can protect the cortical bone against deteriorative effects of ionizing radiation and can be used to improve the material properties of sterilized allografts.     

Effects of gamma radiation on FcepsilonRI and TLR-mediated mast cell activation

Ionizing gamma radiation has several therapeutic indications including bone marrow transplantation and tumor ablation. Among immune cells, susceptibility of lymphocytes to gamma radiation is well known. However, there is little information on the effects of gamma radiation on mast cells, which are important in both innate and acquired immunity. Previous studies have suggested that mast cells may release histamine in response to high doses of gamma radiation, whereas other reports suggest that mast cells are relatively radioresistant. No strong link has been established between gamma radiation and its effect on mast cell survival and activation. We examined both human and murine mast cell survival and activation, including mechanisms related to innate and acquired immune responses following gamma radiation. Data revealed that human and murine mast cells were resistant to gamma radiation-induced cytotoxicity and, importantly, that irradiation did not directly induce beta-hexosaminidase release. Instead, a transient attenuation of IgE-mediated beta-hexosaminidase release and cytokine production was observed which appeared to be the result of reactive oxygen species formation after irradiation. Mast cells retained the ability to phagocytose Escherichia coli particles and respond to TLR ligands as measured by cytokine production after irradiation. In vivo, there was no decrease in mast cell numbers in skin of irradiated mice. Additionally, mast cells retained the ability to respond to Ag in vivo as measured by passive cutaneous anaphylaxis in mice after irradiation. Mast cells are thus resistant to the cytotoxic effects and alterations in function after irradiation and, despite a transient inhibition, ultimately respond to innate and acquired immune activation signals.     

Effects of gamma irradiation on mechanical properties of defatted trabecular bone allografts assessed by speed-of-sound measurement

New sterilization methods for human bone allografts may lead to alterations in bone mechanical properties, which strongly influence short- and medium-term outcomes. In many sterilization procedures, bone allografts are subjected to gamma irradiation, usually with 25 KGy, after treatment and packaging. We used speed-of-sound (SOS) measurements to evaluate the effects of gamma irradiation on bone. All bone specimens were subjected to the same microbial inactivation procedure. They were then separated into three groups, of which one was treated and not irradiated and two were exposed to 10 and 25 KGy of gamma radiation, respectively. SOS was measured using high- and low-frequency ultrasound beams in each orthogonal direction. SOS and Young modulus were altered significantly in the three groups, compared to native untreated bone. Exposure to 10 or 25 KGy had no noticeable effect on the study variables. The impact of irradiation was small compared to the effects of physical or chemical defatting. Reducing the radiation dose used in everyday practice failed to improve graft mechanical properties in this study.     

THE PHYSICIAN AND THE ATOMIC BOMB

Atomic detonations are essentially of two types: contaminating and non-contaminating. The only non-contaminating burst is the high air burst, since it does not result in the contamination of the ground with radioactive bomb residue. This type of burst results in blast, thermal and ionizing radiation injury (often combined in the same patient).The only injurious agent peculiar to atomic warfare is ionizing radiation. With a high air burst these effects are due mainly to gamma rays, and they are no longer present after the first few seconds following the explosion. Although only about 15 per cent of the deaths resulting from this type of burst are likely to be due primarily to ionizing radiations, exposure to the latter may well complicate recovery from trauma.Since there is a latent period of a number of days between the initial and later symptoms and signs of whole body radiation exposure, it does not constitute an emergency and can be treated after the initial period of the disaster has passed.With the detonation of a contaminating burst (a surface, underwater or underground burst) the radii of damage from blast and thermal radiation are considerably less than with a high air burst. Two types of radiation may result from the radioactive fog (base surge) formed after an underwater burst—transit radiation and deposit or continuing radiation. The deposit radiation includes that resulting from inhaled or ingested radioactive material as well as that deposited on clothes or skin. Bomb residue contains material which would localize in bones if it entered the body, and much of it has a long radioactive and biological half-life. It would thus bombard the radiosensitive bone marrow for long periods.Fortunately, the materials which would localize in bone are poorly absorbed from the gastrointestinal tract and lungs.In general radiation injury to a person exposed to a contaminating burst should be reckoned primarily in terms of the penetrating gamma radiation to which he was exposed, rather than in terms of possible internal radiation from ingested or inhaled contaminants.The principles of broad planning, careful triage, decentralization of medical aid, intelligent stockpiling, and the greatest good to the greatest number are to be stressed in medical defense planning.The best appraisal of exposure and its degree of seriousness is, as it is with disease in general, an accurate clinical evaluation by the physician. The tempo of the disease is an important aid in evaluating severity of exposure. The use of the dosimeter in judging the fate of a given individual is, at least at present, of limited value.     

Micronucleus formation in the bone marrow cells of chronically irradiated mice with subsequent acute gamma irradiation]

With the use of the micronuclear test method it has been shown that mice preirradiated with gamma rays at a low dose rate exhibit a decreased frequency of chromosome aberrations induced in bone marrow cells by subsequent acute exposure to gamma radiation as compared to mice not subjected to preliminary irradiation. Such animals have a higher radioresistance with respect to the survival rate. The results obtained suggest the possibility of induction by ionizing radiation, at a low dose rate, of adaptive repair response at the organism level.     

Evaluation of the Effect of a Gamma Irradiated DBM-Pluronic F127 Composite on Bone Regeneration in Wistar Rat

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.     

Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm³. Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm³ and a lesser amount banded at 1.22 to 1.23 g/cm³. Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either ³²PO₄ or ³H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm³) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm³), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.     

Effect of accelerated electron beam on mechanical properties of human cortical bone: influence of different processing methods

Accelerated electron beam (EB) irradiation has been a sufficient method used for sterilisation of human tissue grafts for many years in a number of tissue banks. Accelerated EB, in contrast to more often used gamma photons, is a form of ionizing radiation that is characterized by lower penetration, however it is more effective in producing ionisation and to reach the same level of sterility, the exposition time of irradiated product is shorter. There are several factors, including dose and temperature of irradiation, processing conditions, as well as source of irradiation that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect e-beam irradiation with doses of 25 or 35?kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors, aged from 46 to 54?years, were transversely cut into slices of 10?mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as e-beam irradiation dose (25 or 35?kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis, including all groups, it was found that temperature of e-beam irradiation and defatting had no consistent significant effect on evaluated mechanical parameters of compact bone rings. In contrast, irradiation with both doses significantly decreased the ultimate strain and its derivative toughness, while not affecting the ultimate stress (bone strength). As no deterioration of mechanical properties was observed in the elastic region, the reduction of the energy absorption capacity of irradiated bone rings apparently resulted from changes generated by irradiation within the plastic strain region.     

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells.

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.     

Analysis of biological samples using prompt gamma radions induced by 14.7-MeV neutrons

We investigate a method for determining the elemental composition of biological samples that uses prompt gamma rays induced by 14.7-MeV neutrons. Alpha particles are produced simultaneously with the neutrons, which exit opposite the alpha detector through the vacuum chamber wall. The sample under investigation is irradiated and emits gamma radiations in a spectral energy distribution characteristic of the material. Barium-fluoride (BaF₂) and high-purity germanium (HPGe) gamma detectors view the sample and record the spectrum of gamma radiation.

     

Response kinetics of radiation-induced micronucleated reticulocytes in human bone marrow culture

The frequency of micronucleated reticulocytes (MN-RETs) in the bone marrow or peripheral blood is a sensitive indicator of cytogenetic damage. While the kinetics of MN-RET induction in rodent models following irradiation has been investigated and reported, information about MN-RET induction of human bone marrow after radiation exposure is sparse. In this report, we describe a human long-term bone marrow culture (LTBMC), established in three-dimensional (3D) bioreactors, which sustains long-term erythropoiesis. Using this system, we measured the kinetics of human bone marrow red blood cell (RBC) and reticulocyte (RET) production, as well as the kinetics of human MN-RET induction following radiation exposure up to 6Gy. Human bone marrow established in the 3D bioreactor demonstrated an average percentage of RBCs among total viable cells peaking at 21% on day 21. The average percentage of RETs among total viable cells reached a maximum of 11% on day 14, and remained above 5% by day 28, suggesting that terminal erythroid differentiation was still active. Time- and dose-dependent induction of MN-RET by gamma radiation was observed in the human 3D LTBMC, with peak values occurring at approximately 3 days following 1Gy irradiation. A trend towards delayed peak to 3-5 days post-radiation was observed with radiation doses ≥2Gy. Our data reveal valuable information on the kinetics of radiation-induced MN-RET of human bone marrow cultured in the 3D bioreactor, a synthetic bioculture system, and suggest that this model may serve as a promising tool for studying MN-RET formation in human bone marrow, thereby providing opportunities to study bone marrow genotoxicity testing, mitigating agent effects, and other conditions that are not ordinarily feasible to experimental manipulation in vivo.     

An outbreak of Eastern equine encephalitis virus in free-ranging white-tailed deer in Michigan

Eastern equine encephalitis (EEE) virus has been recognized as affecting horses and humans in the eastern United States for 70 yr. Evidence of exposure with EEE virus has been reported in a variety of free-ranging wild birds and mammals but cases of clinical disease are much less commonly reported. In Michigan, reports of outbreaks of EEE virus in equine species extend back more than a half century. We report diagnosis of EEE virus infection of multiple free-ranging white-tailed deer (Odocoileus virginianus) from three Michigan counties during late summer of 2005. Infection was confirmed in seven of 30 deer collected based on reported neurologic signs and results from immunohistochemistry, polymerase chain reaction, and/or virus isolation. One of the deer also was infected with West Nile virus and an eighth deer had microscopic lesions in the cerebrum consistent with those reported for EEE. To our knowledge, this is the first report of multiple cases of EEE in free-ranging white-tailed deer, and highlights several issues of significance to wildlife managers and public health officials.     

Determining the repair rate for radiation-induced chromatid deletions during the first 24 hours after irradiation

The number of chromatid plus isochromatid deletions present in the testes and bone marrow of the Chinese hamster was measured as a function of time following acute exposure to cobalt-60 gamma irradiation. The number of breaks remaining scorable at any time after irradiation exposure could be represented by a simple exponential equation (N = Noe-kt). The values of No and k, calculated after 100 roentgens of cobalt-60 gamma irradiation, were 1.15 breaks/cell and 0.16/hour, respectively, for the testes, and 0.53 break/cell and 0.14/hour, respectively, for the bone marrow. The average length of time that the breaks remain scorable, 1/k, was found to be 6.3 hours for the testes and 7.1 hours for the bone marrow. By suitable formulation, it was possible to predict the number of breaks present by using varied radiation exposure rates and times after exposure. This formulation could be used for both single and multiple radiation exposures.     

Abundance and temporal distribution of Ornithonyssus sylviarum Canestrini and Fanzago (Acarina: Mesostigmata) in gray catbird (Dumatella carolinensis) nests.

The northern fowl mite, Ornithonyssus sylviarum Canestrini and Fanzago, is a common ectoparasite of wild birds. Despite its ability to transmit eastern equine encephalitis (EEE) virus under laboratory conditions and potential for involvement in the natural EEE virus cycle, we know little about its abundance or temporal distribution in nature. From June to August 2000, we studied the abundance of O. sylviarum in the nests of gray catbirds (Dumatella carolinensis), a reservoir host for EEE virus, at Killbuck Marsh Wildlife Area (KMWA), a known EEE virus focus in Wayne County, Ohio. A total of 7,883 O. sylviarum, including 1,910 adults and 5,973 protonymphs, were recovered from 23 of 26 gray catbird nests collected during various phases of the nesting cycle. We found no association between mite abundance and number of catbird nestlings in successful nests. However, mite abundance increased significantly with date of nest collection and peaked in late July when transmission of EEE virus is likely to occur at KMWA. We therefore suggest that O. sylviarum may contribute to the transmission of EEE virus among gray catbirds at KMWA.