Validation and Substantiation of 25 kGy as Sterilization Dose for Lyophilized Human Amnion Membrane

The validation and substantiation of sterilization dose for lyophilized human amnion membrane by gamma irradiation delivered by Co⁶⁰ source were investigated. The validation experiments were conducted according to ISO 13409 method B. A total of 120 human amnion membranes were collected. Of these, 10 membranes were used for estimation of bioburden and 20 membranes were used for the individual sterility test at verification dose. The average bioburden per product unit with sample item portion (SIP = 1) for lyophilized human amnion membrane was 572 cfu. The verification dose experiments were done at dose of 8.1 kGy and the results of sterility tests showed that human amnion membrane got one positive. Consequently, the sterilization dose of 25 kGy was confirmed and substantiated.     

Validation of Radiation Sterilization Dose for Lyophilized Amnion and Bone Grafts

A limited number of grafts produced in one batch is the main constrain to validate radiation sterilization dose of amnion and bone grafts according to ISO standard. The validation experiments done were according to ISO 13409 with a slight modification in sampling method. The experiments were carried out three times by using 20 samples each, 10 for bio-burden enumeration and 10 for sterility test at verification dose. The average bio-burden with sample item portion (SIP) = 1 for amnion membranes were 98, 50 and 69 cfu respectively and 0 cfu for bone grafts. Verification dose experiments, were done at doses of 2.90kGy for bone grafts and 5.13kGy for amnion grafts and the results of sterility tests showed that amnion grafts got one positive and bone grafts got 0 positive. The results met the requirements of ISO 13409 so that the radiation sterilization dose, at sterility assurance level of 10^-6 was 25kGy for both amnion and bone grafts. Viral contamination was excluded in this experiment.     

Validation of 15 kGy as a radiation sterilisation dose for bone allografts manufactured at the Queensland Bone Bank: application of the VD<Subscript>max</Subscript> 15 method

Background ISO 11137-2006 (ISO 11137-2a 2006) provides a VD_max 15 method for substantiation of 15 kGy as radiation sterilisation dose (RSD) for health care products with a relatively low sample requirement. Moreover, the method is also valid for products in which the bioburden level is less than or equal to 1.5. In the literature, the bioburden level of processed bone allografts is extremely low. Similarly, the Queensland Bone Bank (QBB) usually recovers no viable organisms from processed bone allografts. Because bone allografts are treated as a type of health care product, the aim of this research was to substantiate 15 kGy as a RSD for frozen bone allografts at the QBB using method VD_max 15—ISO 11137-2: 2006 (ISO 11137-2e, Procedure for method VD_max 15 for multiple production batches. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006; ISO 11137-2f, Procedure for method VD_max 15 for a single production batch. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006). Materials 30 femoral heads, 40 milled bone allografts and 40 structural bone allografts manufactured according to QBB standard operating procedures were used. Method Estimated bioburdens for each bone allograft group were used to calculate the verification doses. Next, 10 samples per group were irradiated at the verification dose, sterility was tested and the number of positive tests of sterility recorded. If the number of positive samples was no more than 1, from the 10 tests carried out in each group, the verification was accepted and 15 kGy was substantiated as RSD for those bone allografts. Results The bioburdens in all three groups were 0, and therefore the verification doses were 0 kGy. Sterility tests of femoral heads and milled bones were all negative (no contamination), and there was one positive test of sterility in the structural bone allograft. Accordingly, the verification was accepted. Conclusion Using the ISO validated protocol, VD_max 15, 15 kGy was substantiated as RSD for frozen bone allografts manufactured at the QBB.     

Substantiation of 25 kGy radiation sterilization dose for banked air dried amniotic membrane and evaluation of personnel skill in influencing finished product bioburden

Preparation of amniotic membrane (AM) by air drying method followed by radiation sterilization is simple and valuable approach; sterility and quality of the final AM product are depending on the quality management system at the tissue bank. Validation and substantiation of radiation sterilization dose (RSD) for tissue allografts is an essential step for the development and validation of the standard operating procedures (SOP). Application of SOP is perfectly relying on trained staff. Skills differences among personnel involved in AM preparation could have an effect on microbiological quality of the finished product and subsequently on the RSD required. AM were processed by four different couples of the tissue bank technicians. The AM grafts were randomly selected and subjected to bioburden test to validate and substantiate the 25 kGy RSD. Bioburden test for AM grafts were also useful to evaluate the skill of the tissue bank technicians and thus, to validate the current SOP for air dried AM. Moreover, the effect of placental source on bioburden counts on AM grafts was assessed. Substantiation of the 25 kGy RSD at a sterility assurance level of 10^?1, and sample item portion = 1, was carried out using Method VD _max ²⁵ of the International Organization for Standardization, document no. 11137-2 (ISO in Sterilization of healthcare products—radiation—part 2: establishing the sterilization dose, Method VDmax—substantiation of 25 kGy or 15 kGy as the sterilization dose, International Standard Organization, 2006). The results showed that there were no significant differences in the bioburdens of the four batches (α = 1 %), this means no significant differences in the skill of the four couples of the tissue bank technicians in terms of their ability to process AM according to the air dried AM SOP. The 25 kGy RSD was validated and substantiated as a valid sterilization dose for the AM prepared with the current established SOP at the Biotechnology Research Center experimental tissue bank. The donor’s type of delivery, normal or caesarean, showed no significant effect on the levels of microbial counts on the tested AMs (α = 1 %).     

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.     

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.     

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.     

Structural mechanical properties of radiation-sterilized human Bone-Tendon-Bone grafts preserved by different methods

To avoid the risk of infectious disease transmission from donor to recipient, allografts should be terminally sterilized. In the previous paper (Kaminski et al. in Cell Tissue Bank 10:215–219, 2009) we presented the effect of various methods of preservation (deep fresh freezing, glycerolization, lyophilization), followed by irradiation with different doses of electron beam (EB), on material (intrinsic) mechanical properties of human patellar tendons cut out as for anterior cruciate ligament reconstruction, obtained in failure tensile test. As structural mechanical properties are equally important to predict the behaviour of the graft as a whole functional unit, the purpose of the present paper was to show the results for failure load and elongation, obtained in the same experiment. Paired Bone-Tendon-Bone grafts (BTB) were prepared from cadaveric human patella tendons with both patellar and tibial attachments. They were preserved by deep freezing, glycerolization or lyophilization and subsequently EB-irradiated with the doses of 25, 35, 50 or 100 kGy (fresh-frozen grafts) or a single dose of 35 kGy (glycerolized and lyophilized grafts). Each experimental (irradiated) group was provided with control (non-irradiated), donor-matched group. The specimens from all groups were subjected to mechanical failure tensile test with the use of Instron system in order to measure their structural properties (failure load and elongation). All lyophilized grafts were rehydrated before mechanical testing. In our study we did not observe significant deterioration of structural mechanical properties of BTB grafts processed by fresh-freezing and then terminal sterilized with growing doses of EB up to 100 kGy. In contrast, BTB grafts processed by glycerolization or lyophilization and irradiated with 35 kGy showed significant decrease of failure load. Obtained results suggest that deep-frozen irradiated grafts retain their initial mechanical properties to an extent which does not exclude their clinical application. However, biomechanical investigations constitute only the first step to evaluate the potential clinical usefulness of such allografts and further extensive in vivo studies are needed.     

Revascularization of cranial versus iliac crest bone grafts in the rat

Full-thickness cranial (membranous) and split-thickness iliac crest (endochondral) onlay bone grafts were placed subperiosteally without fixation onto the snout (membranous) and tibia (endochondral) in 30 rats. The animals had been divided into three equal groups in which the bone grafts had been demineralized, autoclaved, or used fresh. Recipient sites were harvested at 7 and 14 days at the snout and 14 days at the tibia, and revascularization was studied utilizing silicone rubber injection and a gridcounting technique. Endochondral grafts were found to have quantitatively greater revascularization than membranous grafts in all three groups at both sites (p less than 0.005). There was generally no statistically significant difference in revascularization between fresh and demineralized grafts, but vessel ingrowth was significantly decreased in autoclaved implants as compared with fresh grafts. Differences in graft architecture are theorized to account for the disparity in revascularization in endochondral and membranous grafts. Angiogenic and chemotactic factors are thought to play a role in the similarities and differences in revascularization among fresh, demineralized, and autoclaved bone.     

Heat resistance of allograft tissue

Lyophilized allograft musculoskeletal tissue is generally intended to be stored at “ambient” or “room” temperature, and usually is kept in climate controlled indoor storage areas. However, there is a question of what temperature extremes tissue may see, especially during transportation, in that these extremes may exceed even the limits of “ambient” conditions. Temperatures may become quite hot, but only for a few hours and only during daytime. Damage from high temperatures, if it occurs, is expected to be evident by damage to the collagen component of bone, soft tissue, and demineralized bone, as well as to the growth factors contained in demineralized bone. If damage is significant, then temperature monitoring requirements for lyophilized allograft tissue might be necessary. To answer this question, a literature review was carried out to look at the short-term temperature resistance of collagen and demineralized bone. Both collagen and the growth factors in demineralized bone show remarkable short term tolerance to elevated temperatures in the dry state, and it was concluded that temperature excursions of 50°C or less, lasting for a few days or less, would not cause any significant deterioration. This means that temperature monitoring also should not be required.     

Radiation Resistance of the Microflora Associated with Amniotic Membranes

Summary Amniotic membrane is widely used in the treatment of burn wounds and ulcers of various etiology. As it comes into contact with open wounds, it needs to be perfectly sterile to avoid the transmission of any disease. Accordingly, amniotic membrane needs to bear a high sterility assurance level (SAL). Conventionally, a radiation dose of 25 kGy is the generally accepted dose for sterilization. But to keep intact the biomechanical and other properties, it is sometimes proposed to use a lower dose without compromising an SAL of 10⁻⁶. The initial microbial contamination level and the radiation resistance of the contaminants determine the dose required for sterilization. The microbial species associated with the amniotic membrane from about 70 different batches were isolated. Twenty-two representative bacterial isolates were characterized and tested for survival in an incremental series of radiation doses from 0.5 to 5.0 kGy. The radiation decimal reduction dose (D₁₀) values for the strains were determined. Relatively higher D₁₀ values were recorded for the gram-positive isolates. The D₁₀ values of microbial isolates ranged from 0.16 to 1.3 kGy, and most resistant Bacillus strain had a D₁₀ value of 2.1 kGy. The radiation dose necessary to achieve an SAL of 10⁻⁶ was calculated based on the D₁₀values of the isolated strains. For a bioburden of 1000 Bacillus organism, the sterilization dose of 18.9 kGy is obtained. However, based on the experimental determination of D₁₀ of the radiation-resistant reference strain Bacillus pumilus, the adequate dose for radiation sterilization is found to be 19.8 kGy if bioburden level of 1000 is granted. The results substantiate that radiation dose of 25 kGy assures sterilization of amniotic membranes with bioburden level of 1000 colony forming units.     

Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses.

In the production of bone grafts intended for transplantation, basic safety measures to avoid the transmission of pathogens are selection and serological screening of donors for markers of virus infections. As an additional safety tool we investigated the effect of gamma irradiation on the sterility of human bone diaphysis transplants and evaluated its impact on the virus safety of transplants. Model viruses were included in the study to determine the dose necessary to achieve a reduction factor for the infectivity titres of at least 4 log(10) at a temperature of -30+/-5 degrees C. The following viruses were used: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), and poliovirus (PV-1), and the following model viruses: pseudorabies virus (PRV) as a model for human herpesviruses, bovine viral diarrhoea virus (BVDV) for HCV, and bovine parvovirus (BPV) for parvovirus B19. A first approach was to determine the D(10) values (kGy) for the different viruses (virus inactivation kinetics: BPV 7.3; PV-1 7.1; HIV-2 7.1; HAV 5.3; PRV 5.3; BVDV <3.0 kGy). Based on these results, inactivation of these viruses was studied in experimentally contaminated human bone transplants (femoral diaphyses). For BPV, the most resistant one of the viruses studied, a dose of approximately 34 kGy was necessary to achieve a reduction of infectivity titres of 4 log(10). We therefore recommend a dose of 34 kGy for the sterilisation of frozen bone transplants.     

Disinfection of human skin allografts in tissue banking: a systematic review report

The use of skin allografts to temporarily replace lost or damaged skin is practiced worldwide. Naturally occurring contamination can be present on skin or can be introduced at recovery or during processing. This contamination can pose a threat to allograft recipients. Bacterial culture and disinfection of allografts are mandated, but the specific practices and methodologies are not dictated by standards. A systematic review of literature from three databases found 12 research articles that evaluated bioburden reduction processes of skin grafts. The use of broad spectrum antibiotics and antifungal agents was the most frequently identified disinfection method reported demonstrating reductions in contamination rates. It was determined that the greatest reduction in the skin allograft contamination rates utilized 0.1 % peracetic acid or 25 kGy of gamma irradiation at lower temperatures.     

Radiation-induced effects on cefotaxime: ESR study

As an alternative to heat and gas exposure sterilization, ionizing radiation is gaining interest as sterilization process for medicinal products. Detection and dosimetry of pharmaceuticals radiosterilization is a growing concern to numerous government regulatory agencies worldwide. In this context, it is necessary to find methods distinguishing between irradiated and non-irradiated pharmaceuticals. In the absence of suitable detection methods, our attention was focused on electron spin resonance (ESR) spectrometry. A third generation cephalosporin, cefotaxime, was chosen as model; this antibiotic is a potential candidate for radiation treatment due to its thermosensitivity. While the ESR spectra of a nonirradiated sample presents no signal, a signal, dependent of the irradiation dose, is found in irradiated samples. The number of free radicals was estimated by comparing the second integral from radiosterilized samples and a diphenylpicrylhydrazyl reference. Estimation of the number of free radicals gives 1.9 × 10²⁰ radicals mol^-1 at 20 kGy. From this result, the G-value (number of radicals (100 eV)^-1) could be estimated to 0.3. Aside from qualitative detection, ESR spectrometry can be used for dose estimation. When quadratic, exponential or bi-exponential functions are applied to the variation of peak to peak amplitude vs. dose, these functions correlate well with the data. However, it is important to notice that linear function correlates well with the data for doses lower than 20 kGy. Since the radiation dose selected must be always based upon the bioburden of the products and the degree of sterility required (EN 552 and ISO 11137) 25 kGy could no longer be accepted as a “routine dose” for sterilizing a pharmaceutical. Doses from 6 kGy (ISO 11137) could be investigated and linear regression would appear to be the least expensive route to follow. The free radicals concentration appeared to not decrease during the 57 days of storage; the number generated during the irradiation allows the detection of radiosterilized cefotaxime up to two years after irradiation.     

Preserved Tissue Allografts in Reconstructive Surgery

Results of treatment with three various kinds of allografts: lyophilized bone, deep frozen bone and cartilage preserved in physiological solution, all of them radiation-sterilized are presented. We believe that this presentation may be helpful in estimating the tissue bank's allografts and in establishing indications and contraindications in the application of allografts in surgery. The ‘indices of coincidence’ were compared in a group of 1014 patients after bone (lyophilized and radiation-sterilized) transplantation. It seems that such a variable as ‘rebuilding of graft’ may be of prognostic value in analysing the results of treatment in this group. The application of frozen and radiation-sterilized allogenic bone grafts for reconstructions is also described. An analysis of the results of treatment in 1125 patients reveals that the use of preserved bone reduces the extent and duration of surgery. Almost total substitution of grafts may be seen in 3–8 months after surgery. Allogenic, preserved cartilage is often used in facial reconstructions of face. Human costal cartilage, preserved in 0.9% NaCl and radiation-sterilized, was used for reconstruction. The patients were examined 24–190 months after surgery (in several clinical units) and results were collected in a special questionnaire by the team that performed surgery. In an analysed group of 437 patients after cartilage transplantation, 42.2% were operated because of posttraumatical changes, 29.0% because of congenital malformations and in 16.7% non-specific inflammations were the cause of reconstructive operations. Malformations were located mainly in the nose (59%), the ear concha (16.5%) and 10.9% were mandible. The results of treatment were compared with ages of patients, diagnosis and the locations of the changes. Very good results were achieved in 33.5% of the patients, and satisfactory in 41.8% of the patients. However, in 19.9% of the patients the result of treatment was unsatisfactory. Correlation between some clinical and biological characteristics and the result of treatment is under discussion. This revised version was published online in July 2006 with corrections to the Cover Date.     

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).     

Lyophilized veins as arterial interposition allografts

Venous allografts were evaluated in two models. Lyophilized allograft veins used as interposition grafts in the infrarenal aorta of the canine were studied and found to be patent at 1 year. Pathologic examination of the grafts revealed mild intimal hyperplasia and persistence of the basic structure of the lyophilized vessel. The ability of venous tissue to elicit an antibody response when transplanted into an allogeneic recipient was studied in the rat using the lymphocyte cytotoxicity assay. Fresh and Me₂SO-cryoprotected frozen veins produced circulating antibody when used as interposition grafts in the infrarenal aorta of the rat. Lyophilized and noncryoprotected frozen veins did not induce measurable antibody. Lyophilized allograft veins are a nonimmunogenic vascular graft material with acceptable long-term patency.     

Considerations in determination of residual moisture in lyophilized demineralized bone matrix: the role of residual moisture analyzers

The objective of this study is to determine whether a residual moisture analyzer (RMA) can be an acceptable instrument for measuring the residual moisture in lyophilized demineralized bone matrix (DBM). Instruments from two different manufacturers with differing configurations and controls were compared: the Ohaus MB45 and Arizona Instrument MAX4000XL. The effects of various factors such as test temperature, drying profile, end point criteria, lift compensation, chamber configuration, and rehydration on residual moisture (RM) are examined. The performance of the RMAs is based on their ability to reproduce RM results obtained by the current standard gravimetric method. RMAs provide reliable, accurate and reproducible results in a number of industries that rely on the determination of RM. We hypothesize that RMAs are suitable for measuring RM in DBM and provide validation study data with optimized settings for these two instruments. Potentially, such studies will provide justification for allowance of this methodology as an acceptable alternative to the current gravimetric method allowed by American Association of Tissue Banks Standards.     

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.     

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.