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.     

Effects of <Superscript>60</Superscript>Co gamma radiation dose on initial structural biomechanical properties of ovine bone—patellar tendon—bone allografts

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose ⁶⁰Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.     

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3–21.8 kGy) and a moderate absorbed dose (24.0–28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.     

The effect of sterilization on the mechanical properties of intact rabbit humeri in three-point bending, four-point bending and torsion

Load bearing bone allografts are used to replace the mechanical function of bone that has been removed or to augment bone that has been damaged in trauma. In order to minimize the risk of infection and immune response, the bone is delipidated and terminally sterilized prior to implantation. The optimal method for bone graft sterilization has been the topic of considerable research. Recently, supercritical carbon dioxide (SCCO₂) treatments have been shown to terminally sterilize bone against a range of bacteria and viruses. This study aimed to evaluate the effect of SCCO₂ treatment compared with two doses of gamma irradiation, on the mechanical properties of whole bone. Paired rabbit humeri were dissected and randomly assigned into either SCCO₂ control, SCCO₂ additive or gamma irradiation at 10 or 25 kGy treatment groups. The bones were mechanically tested in three-point and four-point bending and torsion, with the lefts acting as controls for the treated rights. Maximum load, energy to failure and stiffness were evaluated. This study found that SCCO₂ treatment with or without additive did not alter maximum load, energy to failure or stiffness significantly under any loading modality. Gamma irradiation had a deleterious dose dependant effect, with statistically significant decreases in all mechanical tests at 25 kGy; while at 10 kGy there were reductions in all loading profiles, though only reaching statistical significance in torsion. This study highlights the expediency of SCCO₂ treatment for bone allograft processing as terminal sterilization can be achieved while maintaining the intrinsic mechanical properties of the graft.     

Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods

The secondary sterilisation by irradiation reduces the risk of infectious disease transmission with tissue allografts. Achieving sterility of bone tissue grafts compromises its biomechanical properties. There are several factors, including dose and temperature of irradiation, as well as processing conditions, that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect of gamma 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 gamma 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 it was found that defatting of bone rings had no significant effect on any mechanical parameter studied, whereas irradiation with both doses decreased significantly the ultimate strain and its derivative toughness. The elastic limit and resilience were significantly increased by irradiation with the dose 25?kGy, but not 35?kGy, when the time of irradiation was longer. Additionally, irradiation at ambient temperature decreased maximum load, elastic limit, resilience, and ultimate stress. As strain in the elastic region was not affected, decreased elastic limit resulted in lower resilience. The opposite phenomenon was observed in the plastic region, where in spite of the lower ultimate stress, the toughness was increased due to the increase in the ultimate strain. The results of our study suggest that there may be an association between mechanical properties of bone tissue grafts and the damage process of collagen structure during gamma irradiation. This collagen damage in cortical bone allografts containing water does not depends on the temperature of irradiation or defatting during processing if dose of gamma irradiation does not exceed 35?kGy.     

Sterilization with electron beam irradiation influences the biomechanical properties and the early remodeling of tendon allografts for reconstruction of the anterior cruciate ligament (ACL)

Although allografts for anterior cruciate ligament (ACL) replacement have shown advantages compared to autografts, their use is limited due to the risk of disease transmission and the limitations of available sterilization methods. Gamma sterilization has shown detrimental effects on graft properties at the high doses required for sufficient pathogen inactivation. In our previous in vitro study on human patellar tendon allografts, Electron beam (Ebeam) irradiation showed less detrimental effects compared to gamma sterilization (Hoburg et al. in Am J Sports Med 38(6):1134-1140, 2010). To investigate the biological healing and restoration of the mechanical properties of a 34?kGy Ebeam treated tendon allograft twenty-four sheep underwent ACL replacement with either a 34?kGy Ebeam treated allograft or a non-sterilized fresh frozen allograft. Biomechanical testing of stiffness, ultimate failure load and AP-laxity as well as histological analysis to investigate cell, vessel and myofibroblast-density were performed after 6 and 12?weeks. Native sheep ACL and hamstring tendons (HAT, each n?=?9) served as controls. The results of a previous study analyzing the remodeling of fresh frozen allografts (n?=?12) and autografts (Auto, n?=?18) with the same study design were also included in the analysis. Statistics were performed using Mann-Whitney U test followed by Bonferroni-Holm correction. Results showed significantly decreased biomechanical properties during the early remodeling period in Ebeam treated grafts and this was accompanied with an increased remodeling activity. There was no recovery of biomechanical function from 6 to 12?weeks in this group in contrast to the results observed in fresh frozen allografts and autografts. Therefore, high dose Ebeam irradiation investigated in this paper cannot be recommended for soft tissue allograft sterilization.     

Validating a Low Dose Gamma Irradiation Process for Sterilizing Allografts Using ISO 11137 Method 2B

This paper describes the validation of an allograft sterilization method specifically designed for the processing methods used at AlloSource in Centennial, CO. The methods used for this validation followed ISO Standard 11137, Method 2B. Three hundred allografts, collected from three defined production batches were dosed using a series of five incremental doses, beginning at 1 kGy and increasing by 1 kGy until 5 kGy was achieved. Following sterilization dosing, each allograft test article was analyzed using a sterility test to identify any viable microorganisms. The number of positive sterility samples was used to calculate the verification dose (1.27 kGy), which was then verified by an additional batch of 100 allografts. The results from this validation indicate that sterility (10⁻⁶ SAL) on human allograft tissue using gamma ⁶⁰Co radiation can be achieved when a dose of at least 9.2 kGy is employed.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Irradiation as a Safety Procedure in Tissue Banking

The Central Tissue Bank in Warsaw was established in 1963 and since then ionising radiation has been routinely applied to sterilise tissue grafts. Connective tissue grafts such as bone, cartilage, tendons, sclera, pericardium, skin, acellular dermis and amnion irradiated with a dose of 35 kGy in a ⁶⁰Co source and/or with an electron beam 10 MeV accelerator are prepared in our Tissue Bank and two other multi-tissue banks operating in Poland. Over 250,000 radiation-sterilised tissue grafts have been prepared and used in hospitals throughout Poland and no infectious disease transmission or other adverse post-transplantation reactions have been reported up to today. It should be kept in mind however, that high doses of ionising radiation can evoke numerous chemical and physical changes that may affect the biological quality of tissue allografts. Therefore, interdisciplinary research has been undertaken at the Central Tissue Bank in Warsaw to establish the origin and stability of free radicals and other paramagnetic entities induced by irradiation in bone. The effects of various preservation procedures (e.g. lyophilisation, deep-freezing) and irradiation conditions (doses, temperature of irradiation) on the osteoinductive potential and mechanical properties of bone and on the degradation of collagen, a major constituent of all connective tissue grafts, have been also studied. The results of these studies indicate that radiation-induced changes can be diminished by modification of tissue preservation methods and that, to some extent, it is possible to reduce undesired radiation-induced damage to the tissue grafts.     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.     

Validation of Radiation Dose Received by Frozen Unprocessed and Processed Bone during Terminal Sterilisation

Fresh frozen femoral heads (FH) and frozen processed bone (FP) are widely used as a source of allograft bone. The FP bone and some of the FH are terminally sterilised by the National Blood Service Tissue Services (NSBTS), via application of a minimum 25 kGy gamma radiation dose. To comply with the Guidelines for the Blood Transfusion Services in the United Kingdom (2002), frozen musculoskeletal tissue must be maintained below −40 °C during storage and transit. In practice, NBSTS stores bone long-term in −80 °C freezers. During transport for irradiation, a temperature of circa −79 °C is maintained by packing the bone in dry ice. An evaluation of the radiation dose received by bone has previously been made via dosimeters located within the tissue and dry ice, however, some evidence suggests that low temperature can influence the accuracy of the dosimeter readings. The aim of this study was to determine the actual radiation dose received by FH and FP bone during the irradiation process. This was accomplished by comparing radiation dose readings from dosimeters placed in dry ice with dosimeters placed in a dry ice substitute of similar dimensions and density i.e., polytetrafluoroethylene (PTFE) at ambient temperature. New packing formats were developed for both FH and FP bone such that 15 FH or 3 kg of FP bone could be irradiated in one transport box at any given time in a standardised fashion. The data show that low temperature consistently increased dosimeter readings 10–27%, and that radiation dose always fell within the range of 25–40 kGy (FH = 25.1–35.7 kGy; FP bone = 25.2–32.4 kGy).     

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.     

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.     

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.     

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.     

Effect of x-rays and gamma radiations on the bone mechanical properties: literature review

The bone auto grafting, isografting, allografting and xenografting are used for defective bone replacement or treatment in almost all living species. The X-ray and Gamma (electromagnetic radiation) sterilization performed on the donor bone graft to prevent toxicity or migration of virus/bacterial infections from donors to reciver. Conversely, X-ray and Gamma radiation deteriorates the bone mechanical properties and bone become more susceptible to fracture. Fracture toughness as well as other mechanical properties of bone change with these radiations. In this literature review the effect of the X-rays and Gamma radiation on bone mechanical properties are discussed. All relevant literature was reviewed. After reviewing the literature only the research relating to the effect of X-rays and Gamma radiations on bone mechanical properties are included. Literature studies showed significant effect of the X-rays and Gamma radiations on the mechanical properties of the bones. In some studies the differences exists on the doses of radiations which were discussed in this study. The high energetic electromagnetic radiation (X-rays and Gamma radiations) changed/modify the collagen network of the bone, which reduced the mechanical properties of bone; however these changes depend on the radiation dose.     

Gamma radiation resistance of Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.     

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.     

Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and ⁶⁰Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and ⁶⁰Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.     

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage ϕ×174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated >6.1log₁₀ of the model virus, and gamma irradiation (at 25–40 kGy and applied to frozen allografts) inactivated 3.6–4.0log₁₀ of the model virus and >4log₁₀ of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.