A. Hoburg S. Keshlaf T. Schmidt M. Smith U. Gohs C. Perka A. Pruss S. Scheffler

Transcription

1 Cell Tissue Bank (2015) 16: DOI /s x ORIGINAL PAPER High-dose electron beam sterilization of soft-tissue grafts maintains significantly improved biomechanical properties compared to standard gamma treatment A. Hoburg S. Keshlaf T. Schmidt M. Smith U. Gohs C. Perka A. Pruss S. Scheffler Received: 18 January 2014 / Accepted: 5 July 2014 / Published online: 19 July 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Allografts have gained increasing popularity in anterior cruciate ligament (ACL) reconstruction. However, one of the major concerns regarding allografts is the possibility of disease transmission. Electron beam (Ebeam) and Gamma radiation have been proven to be successful in sterilization of medical products. In soft tissue sterilization high dosages of gamma irradiation have been shown to be detrimental to biomechanical properties of grafts. Therefore, it was the objective of this study to compare the biomechanical properties of human bone-patellar tendon-bone (BPTB) grafts after ebeam with standard A. Hoburg (&) T. Schmidt C. Perka Department for Orthopaedic Surgery and Traumatology, Center for Muskuloskeletal Surgery, Sports Medicine and Arthroscopy Service, Charité University Medicine Berlin, Charitéplatz 1, Berlin, Germany arnd.hoburg@charite.de S. Keshlaf A. Pruss Institute for Transfusion Medicine (Tissue Bank), Charité University Medicine Berlin, Berlin, Germany M. Smith German Institute for Cell and Tissue Replacement (DIZG), Berlin, Germany U. Gohs Leibniz-Institute of Polymer Research Dresden, Hohe Straße 6, Dresden, Germany S. Scheffler Chirurgisch Orthopaedischer PraxisVerbund (COPV), Breitenbachplatz 8, Berlin, Germany gamma irradiation at medium (25 kgy) and high doses (34 kgy). We hypothesized that the biomechanical properties of Ebeam irradiated grafts would be superior to gamma irradiated grafts. Paired 10 mm-wide human BPTB grafts were harvested from 20 donors split into four groups following irradiation with either gamma or Ebeam (each n = 10): (A) Ebeam 25 kgy, (B) Gamma 25 kgy, (C) Ebeam 34 kgy (D) Gamma 34 kgy and ten non-irradiated BPTB grafts were used as controls. All grafts underwent biomechanical testing which included preconditioning (ten cycles, 0 20 N); cyclic loading (200 cycles, N) and a load-to-failure (LTF) test. Stiffness of non-irradiated controls (199.6 ± 59.1 N/mm) and Ebeam sterilized grafts did not significantly differ (152.0 ± 37.0 N/mm; ± 58.0 N/mm), while Gamma-irradiated grafts had significantly lower stiffness than controls at both irradiation dosages (25 kgy: ± 45.4 N/mm; 34 kgy: ± 58.2 N/mm) (p \ 0.05). Failure loads at 25 kgy were significantly lower in the gamma group (1,009 ± 400 N), while the failure load was significantly lower in both study groups at high dose irradiation with 34 kgy (Ebeam: 1,139 ± 445 N, Gamma: 1,073 ± 617 N) compared to controls (1,741 ± 304 N) (p \ 0.05). Creep was significantly larger in the gamma irradiated groups (25 kgy: 0.96 ± 1.34 mm; 34 kgy: 1.06 ± 0.58 mm) than in the Ebeam (25 kgy: 0.50 ± 0.34 mm; 34 kgy: 0.26 ± 0.24 mm) and control (0.20 ± 0.18 mm) group that did not differ significantly. Strain difference was not different between either control or study

2 220 Cell Tissue Bank (2015) 16: groups (controls: 1.0 ± 0.03; Ebeam 34 kgy 1.04 ± 0.018; Gamma 34 kgy 1.0 ± 0.028; 25 kgy: 1.4 ± 2,0; 34 kgy: 1.1 ± 1.1). The most important result of this study was that ebeam irradiation showed significantly less impairment of the biomechanical properties than gamma irradiation. Considering the results of this study and the improved control of irradiation application with electronic beam, this technique might be a promising alternative in softtissue sterilization. Keywords Allograft ACL reconstruction Sterilization Ebeam Gamma irradiation Introduction Allografts have gained increasing popularity in anterior cruciate ligament (ACL) reconstruction. This is reflected by an increase of allograft use from 2 % between 1986 and 1996 to 14 % from 1996 to 2001 Bach, Aadalen Bach et al. (2005). In 2003, 1.2 million bone and soft tissue allografts were transplanted in the US McAllister et al. (2007). However, one of the major concerns regarding allografts is the possibility of disease transmission. Current tissue banking regulations for procurement and processing of soft tissue allografts include intensive screening of donor history, aseptic handling, detection assays and terminal sterilization (McAllister et al. 2007). Electron beam (Ebeam) and Gamma radiation have been proven to be successful in sterilization of medical products (Russell 1992). Currently, Gamma irradiation is the most frequently used sterilization procedure for soft-tissue allografts in ACL surgery. The International Atomic Energy Agency (IAEA) recommends an irradiation dose of 25 kgy, which is effective for inactivation of bacteria, but higher doses are required for inactivation of more resistant pathogens as HIV or mold spores (IAEA 1990). For industrial sterilization of medical products the sterility assurance level (SAL) was introduced, recommending a reduction of infectious titres by 6 log 10. This means, that the probability of a viable microorganism being present after sterilization is one in a million. However, different levels of contamination exist in human tissue. Also, it is difficult to determine the actual bioburden of each tissue. Thus, sterilization of biological tissue needs to be effective for reduction of all pathogens possibly present. Therefore, radiation doses of 34 kgy have been recommended, leading to a reduction of parvovirus B19 (which is the most resistant virus to ionization) by at least 4 log 10 Pruss et al. (2002). However, higher doses of radiation have been shown to result into modifications of the microscopic and ultrastructural appearance of tendons and ligaments (Salehpour et al. 1995). This is mainly caused by the release of free radicals, which lead to degradation of collagen fibers and breakage of covalent bonds, leading to significant deterioration of biomechanical properties of the respective grafts (Bailey et al. 1964). Several studies have shown that gamma radiation doses beyond 20 kgy result into significant loss of mechanical stability at time zero (Salehpour et al. 1995; Anderson et al. 1992; Currey et al. 1997; Fideler et al. 1995; Gibbons et al. 1991; Rasmussen et al. 1994; Yusof 2000). Ebeam is an ionizing radiation analog to gamma radiation, which is characterized by its good penetrability, high effectiveness, lack of heat induction during the sterilization procedure or pressure differences and diffusion barriers typically associated with chemical sterilization methods (Yusof 2006). Also, it allows sterilization in closed wrappings, thus eliminating the chance of recontamination during packaging. Unlike gamma radiation, where ionization occurs by photon emission, accelerated electrons of specific mass and charge cause Ebeam irradiation. Hence, their penetrability depends on their energy, atomic number of the absorbing product and its density leading to differences in their interaction with tissue compared to gamma radiation. Their sterilization efficiency is comparable, especially for soft tissue irradiation, as the inferior penetration depth of Ebeam only becomes evident at products with a higher density or thickness. An important advantage of Ebeam irradiation is a substantially improved control of dose application and processing speed and accuracy of the actual sterilization procedure compared to gamma irradiation (Yusof 2006; Dziedzic-Goclawska et al. 2005). Also, it has been shown that the addition of CO 2 with Ebeam sterilization at low temperatures showed a protective effect on tissue of non-human materials (Klose 2006). Surprisingly, considerably less

3 Cell Tissue Bank (2015) 16: Fig. 1 Photo of a human bone patellar tendon bone transplant before mechanical testing literature exists on the impact of Ebeam irradiation of soft-tissue allografts. Therefore, it was the objective of this study to compare the biomechanical properties of human BPTB grafts after Ebeam irradiation with standard gamma irradiation at medium (25 kgy) and high doses (34 kgy). We hypothesized that the biomechanical properties of Ebeam irradiated grafts would be superior to gamma irradiated grafts. Methods Paired 10 mm-wide human BPTB grafts were harvested from 20 donors split into four groups following irradiation with either gamma or Ebeam (each n = 10): (A) Ebeam 25 kgy, (B) Gamma 25 kgy, (C) Ebeam 34 kgy (D) Gamma 34 kgy (Fig. 1). Ten non-irradiated BPTB grafts of identical dimensions were used as controls. For Ebeam irradiation all grafts were individually packed in CO 2 -filled gas-impermeable sterilization bags at an authorized sterilization facility (DIZG, Berlin Germany) and placed on a height-adjusted stage in a dry ice-filled polystyrene box, such that the sterilization process was conducted at approximately -70 and dose could be very tightly controlled (Fig. 2). For gamma irradiation the same packing process was performed without CO 2 Addition. The sterilization process was conducted at Gamma service Produktbestrahlung GmbH (Radeberg, Germany). Mean duration of Ebeam irradiation was 30 s and 16.5 h for gamma irradiation. After sterilization, all grafts were stored in polystyrene boxes at -70 C until the day of mechanical testing. Maximum time of storage was 10 days. All grafts were thawed at room temperature and both femoral and tibial bones were potted in polymethyl methacrylate and mounted on aluminium clamps for fixation on a material testing machine (model 1455; Zwick GmbH, Ulm, Germany) (Fig. 3). The biomechanical testing procedure included: preconditioning (10 cycles, 0 20 N); cyclic loading (200 cycles, N) and a load-to-failure (LTF) test. The strain rate was 150 mm/min. Graft motion during cyclic loading was tracked with an infrared motion analysis system (MacReflex, Qualisys Inc.) (Figs. 3, 4). For analysis of graft motion, four circular reflective tape markers were glued to the tendon surface; two in the midsubstance and two at the bony insertions sites of the grafts. An additional marker was attached to each clamp. The infrared motion analysis system Fig. 2 schematic overview of the sterilization process

4 222 Cell Tissue Bank (2015) 16: Fig. 3 Testing setup with the graft embedded in polymethylmethacrylate and fixated in aluminium clamps for mechanical testing. For measurement with the MacReflex optical measurement system 6 markers (white squares) are attached to the graft and clamp for optical measurement of graft motion consisted of two high-speed cameras (Fig. 4) that measured motion along all three axes. Our preliminary testing showed that out of plane motion was minimal, therefore we only recorded motion along the longitudinal axis of the testing machine between the reflective tape markers attached to the graft s insertion sites. Prior to each test, a special calibration frame was used to calibrate the system, which provided a precision of B0.1 mm for longitudinal graft motion. Customized software (MacReflex, Qualisys Inc.) was used to record and transfer these data to Microsoft Excel Ò software for final data processing. This setting allowed for analysis of overall graft motion during the cyclic loading test. Overall strain and cyclic elongation response were calculated from data obtained with the motion analysis system during cyclic loading testing. We reported strain difference, which was defined as the difference in length at 200 N between the 200th and 1st cycle divided by the initial length of the graft in percent [Strain difference: (L 200 L 1 )/L ]. Overall strain was measured at the last cycle as follows: (L 200 L 0 )/L L 0 was measured as the distance between both markers attached to the respective insertion sites of the patellar tendon at the last cycle of preconditioning at 20 N. Cyclic elongation Fig. 4 photo of the testing setup. The Zwick testing machine can be observed on the right, cameras and computer are part of the optical measurement system (MacReflex)

5 Cell Tissue Bank (2015) 16: response was calculated as the difference in elongation at 200 N between the 200th and first cycle (Cyclic elongation response: Elong 200 Elong 1 ). Structural properties, such as failure load and displacement at failure were recorded and stiffness was derived from these values. Failure loads were defined as the highest load of the linear load-elongation curve, which always corresponded to intratendinous rupture or tendon peal off the tibial insertion site, which was considered graft failure. A more detailed description of the biomechanical testing setup has been previously published (Scheffler et al. 2005). One-way ANOVA and the Bonferroni post hoc test were used to test for significant differences in biomechanical properties between the groups. The level of significance was set at p \ Results Stiffness of non-irradiated controls (199.6 ± 59.1 N/ mm) and Ebeam sterilized grafts did not significantly differ at 25 kgy (152.0 ± 37.0 N/mm) and 34 kgy (192.8 ± 58.0 N/mm), while Gamma irradiated grafts had significantly lower stiffness than controls at both irradiation dosages (25 kgy: ± 45.4 N/mm; 34 kgy: ± 58.2 N/mm) (p \ 0.05). Failure loads at 25 kgy were significantly lower in the gamma group (1,009 ± 400 N), while the failure load was significantly lower in both study groups at high dose irradiation with 34 kgy (Ebeam: 1,139 ± 445 N, Gamma: 1,073 ± 617 N) compared to controls (1,741 ± 304 N) (p \ 0.05) (Table 1). Creep was significantly larger in the gamma irradiated groups (25 kgy: 0.96 ± 1.34 mm; 34 kgy: 1.06 ± 0.58 mm) than in the Ebeam (25 kgy: 0.50 ± 0.34 mm; 34 kgy: 0.26 ± 0.24 mm) and control (0.20 ± 0.18 mm) group that did not differ significantly. Strain difference was not different between either control nor study groups (controls: 1.0 ± 0.03; Ebeam 34 kgy 1.04 ± 0.018; Gamma 34 kgy 1.0 ± 0.028; 25 kgy: 1.4 ± 2,0; 34 kgy: 1.1 ± 1.1) (Table 2). Discussion Aim of the present study was to compare Ebeam and gamma irradiation at medium and high dosages and evaluate their impact on the biomechanical properties of BPTB grafts in vitro. The most important result of this study was that Ebeam irradiation showed significantly less impairment of the biomechanical properties than gamma irradiation. However, both gamma and Ebeam irradiations of bone have been shown to lead to decreased mechanical properties in vitro Nguyen et al. (2007a, b). Dziedzic- Goclawska et al. Compared lyophilized and fresh whole femurs obtained from WAG male rats irradiated at doses of 25, 35 and 50 kgy for their maximum force needed to break the bone. The study showed a dose dependent decrease in strength (43, 57 and 64 % for lyophilized and 17, 25 and 34 % for fresh samples respectively) (Dziedzic-Goclawska 2000). This dose dependent deterioration has also been shown for gamma irradiation in soft tissue allografts (Salehpour et al. 1995). In our study, stiffness of irradiated grafts was lower in gamma irradiated grafts than in Ebeam and non irradiated controls. Even though no statistical significance could be shown for the comparison of both types of irradiation, gamma irradiation resulted into significantly reduced stiffness at 25 and 34 kgy compared to non-irradiated grafts, while no differences were found comparing Ebeam irradiated and non-irradiated allografts. Rasmussen et al. (1994). evaluated the impact of 40 kgy gamma irradiation on BPTB-grafts and found a significant reduction of stiffness values. In this study, this difference was already significant at a dosage of 25 kgy with gamma irradiation. This is consistent with the data of Salehpour et al. (1995), who reported a significant decrease of stiffness with increasing doses of gamma radiation. In contrast to Seto et al. (2008), who found similar reductions in failure loads, our study showed a trend towards less affection in Ebeam irradiation compared to gamma, while non significant. This may be due to the different tendons (human BPTB vs. rabbits Achilles tendon), as well as the different radiation doses 25 and 34 kgy versus 25 and 50 kgy) used. A significant decrease in failure loads at high doses of radiation has been previously reported, which is consistent with our results (Fideler et al. 1995; Mae et al. 2003). In this study, failure loads of Ebeam sterilized grafts were significantly reduced at 34 kgy compared to controls, while Gamma irradiated grafts already showed a significant deterioration of failure loads at 25 and 34 kgy,

6 224 Cell Tissue Bank (2015) 16: suggesting a less detrimental effect of Ebeam irradiation compared to gamma treatment. Creep showed to be significantly larger in gamma irradiated grafts, again suggesting tissue strength to be more affected by gamma than Ebeam irradiation. Co 2 - radical ions have been proposed as a possible cause for the detrimental effects of sterilization procedures. Jastrzebska et al. (2013). measured Co 2 - radical ions in either Ebeam or Gamma irradiated bone and found a dose dependent increase of these ions at ambient temperatures and a significantly lower amount in Ebeam compared to gamma irradiation. However, no marked difference was seen under deep frozen conditions with radical amounts being slightly higher in Ebeam irradiated grafts. Therefore, radical formation might only be one of many changes caused by radiation, as in this study irradiation was performed in deep frozen samples, which were still being less affected than following gamma irradiation Kaminski et al. (2012a, b) also showed that there are no significant differences in biomechanical properties of compact bone rings during Ebeam or Gamma irradiation at ambient temperature or on dry ice in bone. However, ultimate strain significantly decreased at 25 and 35 kgy in both irradiation forms and they proposed collagen damage as a major reason of the detrimental effect on tissue mechanics. Therefore deep freezing of samples as used during the sterilization process in this study should not account for differences seen between Ebeam and Gamma irradiation. Terminal sterilization of soft tissue allografts can ensure tissue banks of higher allograft safety, thus increasing the acceptance of allograft use in surgeons and patients. Modality and dose of sterilization is an ongoing matter of debate. Currently tissue banks use different sterilization procedures, including antibiotic solutions, alcohol, hydrogen peroxide. However, terminal inactivation of all pathogens cannot be achieved with these procedures as these procedures often do not penetrate the whole tissue and have no effect on viral inactivation (Eastlund 2006). Therefore gamma irradiation with doses between 10 and 30 kgy in combination with the aforementioned or as a sole procedure is the current standard procedure. Gamma irradiation has been proven to provide effective sterilization with a high penetration depth up to 40 cm through the density of water. In comparison Ebeam possesses lower penetrability (0.35 cm/mev), which is approximately 8 cm at the density of water. Advantage of Ebeam irradiation is a higher processing speed, which is only seconds compared to several hours in gamma irradiation, which might not only reduce tissue damage, but also provide an economical advantage. Also a more accurate control of the applied dose is inherent with Ebeam irradiation. This is due to the gamma factor, which characterizes the dose deviation of irradiated products due to the shielding effects caused by packing position and products of different densities in the same irradiation procedure. This factor is needed to calculate the applied dosage for gamma irradiation (Dmin = desired dosage 9 dosimeter error 9 gamma factor 9 overdose factor 9 gamma factor 9 dosimeter error). In Ebeam irradiation there is no gamma factor, as each product is directly exposed to irradiation, which reduces the equation to Dmin = desired dosage 9 dosimeter error 9 overdose factor 9 dosimeter error. The dosimeter error is defined as the error occurring due to measurement deviations and is approximately The overdose factor is defined as the quotient of maximal and minimal applied dose (Dmax/Dmin). This factor is dependent on thickness and density of irradiated products and is about 1 for tendons and 1.13 for bone. Hence, when calculating the desired dosage deviation for gamma irradiation, this value is much larger than for Ebeam as is exemplary shown for 34 kgy (Ebeam kgy vs. gamma kgy). Also, the reproducibility of the applied dosage is greater in Ebeam irradiation as the packaging and sterilization setup allows for direct product exposure without shielding effects by other products, that is typically existent in gamma irradiation (Fig. 2). This factor becomes more important when products of different densities are irradiated at once, as it is common in most irradiation facilities. At Gamma service GmbH, where the sterilization in this study has been performed, only products of the same density are sterilized together. Hence, differences found in this study might be more pronounced, if product density is not considered at the time of irradiation. There are some limitations to this study. Only the time zero situation after sterilization was evaluated. Thiscannot predict postimplantation behaviour of grafts, as their remodeling process was not investigated. Despite this, our study shows that the impairment of

7 Cell Tissue Bank (2015) 16: Table 1 Mechanical Properties of electron beam and gamma irradiated BPTB allografts with controls biomechanical properties of soft-tissue allografts at medium and high irradiation doses of 25 kgy and 34 kgy is substantially reduced with the Ebeam procedure compared to standard gamma treatment. Considering the results of this study and the improved control of irradiation application with electronic beam, this technique might be a promising alternative in soft-tissue sterilization. However, a significant reduction of failure strength at high dosages has to be conceded with electronic beam irradiation, too. Therefore, in future studies it is important to gain better understanding of the underlying processes that affect soft-tissue strength and to eventually eliminate these adverse effects. References Stiffness (N/ mm) Max. force (N) Creep (mm) Controls ± ,741 ± ± 0.18 Gamma ± ,009 ± ± 1.34 (25 kgy) Ebeam ± ,177 ± ± 0.34 (25 kgy) Gamma ± ,073 ± ± 0.58 (34 kgy) Ebeam (34 kgy) ± ,139 ± ± 0.24 Table 2 Differences in strain behaviour among the ebeam, gamma and controls Strain 200 (%) Strain 1 (%) Strain difference (%) Controls 3.3 ± ± ± 0.3 Gamma 6.2 ± ± ± 2.0 (25 kgy) Ebeam 7.0 ± ± ± 0.5 (25 kgy) Gamma 5.5 ± ± ± 1.1 (34 kgy) Ebeam (34 kgy) 4.6 ± ± ± 0.4 Anderson MJ, Keyak JH, Skinner HB (1992) Compressive mechanical properties of human cancellous bone after gamma irradiation. J Bone Joint Surg Am 74(5): Bach BR Jr et al (2005) Primary anterior cruciate ligament reconstruction using fresh-frozen, nonirradiated patellar tendon allograft: minimum 2-year follow-up. Am J Sports Med 33(2): Bailey AJ, Rhodes DN, Cater CW (1964) Irradiation-induced crosslinking of collagen. Radiat Res 22: Currey JD et al (1997) Effects of ionizing radiation on the mechanical properties of human bone. J Orthop Res 15(1): Dziedzic-Goclawska, A.(2000), Application of ionising radiation to sterilise connective tissue allografts. Radiation and Tissue Banking, ed. P.G.O. (ed.) Phillips G.O. (ed.) Dziedzic-Goclawska A et al (2005) Irradiation as a safety procedure in tissue banking. Cell Tissue Bank 6(3): Eastlund T (2006) Bacterial infection transmitted by human tissue allograft transplantation. Cell Tissue Bank 7(3): Fideler BM et al (1995) Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts. Am J Sports Med 23(5): Gibbons MJ et al (1991) Effects of gamma irradiation on the initial mechanical and material properties of goat bonepatellar tendon-bone allografts. J Orthop Res 9(2): IAEA (1990), IAEA guidelines for industrial radiation sterilization of disposable medical products (Cobalt-60 Gamma Irradiation) Jastrzebska, A., et al. (2013), Effect of gamma radiation and accelerated electron beam on stable paramagnetic centers induction in bone mineral: influence of dose, irradiation temperature and bone defatting. Cell Tissue Bank Kaminski A et al (2012a) Effect of accelerated electron beam on mechanical properties of human cortical bone: influence of different processing methods. Cell Tissue Bank 13(3): Kaminski A et al (2012b) Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods. Cell Tissue Bank 13(3): Klose, B. (2006). A process for the preparation of a package comprising a sterilized bulk of a drug substance, and a package comprising a sterilized bulk of a penicillin Mae T et al (2003) Effect of gamma irradiation on remodeling process of tendon allograft. Clin Orthop Relat Res 414: McAllister DR et al (2007) Allograft update: the current status of tissue regulation, procurement, processing, and sterilization. Am J Sports Med 35(12): Nguyen H, Morgan DA, Forwood MR (2007a) Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics. Cell Tissue Bank 8(2): Nguyen H, Morgan DA, Forwood MR (2007b) Sterilization of allograft bone: is 25 kgy the gold standard for gamma irradiation? Cell Tissue Bank 8(2):81 91 Pruss A et al (2002) Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses. Biologicals 30(2): Rasmussen TJ et al (1994) The effects of 4 Mrad of gamma irradiation on the initial mechanical properties of bonepatellar tendon-bone grafts. Arthroscopy 10(2): Russell, A. (1992), Principles, practice of disinfection, preservation and sterilization. Principles, Practice of Disinfection,

8 226 Cell Tissue Bank (2015) 16: Preservation and Sterilization. Oxford, UK: Blackwell Scientific Publications Salehpour A et al (1995) Dose-dependent response of gamma irradiation on mechanical properties and related biochemical composition of goat bone-patellar tendon-bone allografts. J Orthop Res 13(6): Scheffler SU et al (2005) Biomechanical comparison of human bone-patellar tendon-bone grafts after sterilization with peracetic acid ethanol. Cell Tissue Bank 6(2): Seto A, Gatt CJ Jr, Dunn MG (2008) Radioprotection of tendon tissue via crosslinking and free radical scavenging. Clin Orthop Relat Res 466(8): Yusof N (2000) Irradiation for sterilising tissue grafts for viral inactivation. Malays J Nucl Sci 18(1):23 35 Yusof, N.(2006), Radiation in Tissue Banking - Basic Science and Clinical Applications of Irradiated Tissue Allografts, A. Nather (ed), Singapore: World Scientific Publishing Co. Ptc. Ltd. 561