Response of bone subjected to optimized high dose irradiation

Allograft tissues are used in over one million musculoskeletal procedures per year. Consequently, it is crucial tissue banks use procedures to militate against allograft associated bacterial and viral infections. Recent studies have identified an important pathogen inactivation technology for musculoskeletal allografts that utilizes high-dose gamma irradiation (50 kGy) under controlled conditions. A total dose of 50 kGy assures that the current standard for medical devices for a microbial sterility assurance level of 10(- 6) is met. Furthermore, the pathogen inactivation technology results in a greater than four log inactivation of enveloped and nonenveloped viruses. Efficacious clinical outcome from musculoskeletal allografts exposed to this innovative sterilization procedure will require that there is no performance decrement in the allograft's biological properties. Therefore, to validate this objective, we executed a study focusing on remodeling and osteoconduction of bone allografts treated with a high dose of gamma irradiation (50 kGy), radioprotectants and well-defined operating parameters of temperature and water content. A rabbit calvarial model was used to test the hypothesis that remodeling and osteoconduction of allogeneic bone treated with the new pathogen inactivation technology would be equivalent to nontreated allogeneic bone. Results indicated treated bone allografts were comparable to nontreated allografts. We conclude, therefore, that based on this outcome and other reports, that high doses of gamma irradiation under optimized conditions designed to reduce free radical damage to tissue will provide safer allografts.     

The effect of sterilization methods on the osteoconductivity of allograft bone in a critical-sized bilateral tibial defect model in rabbits

Clinically, allogeneic bone graft is used extensively because it avoids the donor site morbidity associated with autograft. However, there are concerns over the optimal sterilization method to eliminate immunological risks whilst maintaining the biological efficacy of the graft. This study compared the effect of Supercritical fluid (SCF) treatment and gamma irradiation at 25 kGy on the osteoconductivity of allograft bone in a bilateral critical sized defect rabbit model. Osteoconductivity was evaluated at 2 and 4 weeks using X-ray, CT, histology (qualitative and quantitative) and immunohistochemistry (Alkaline Phosphatase and Cathepsin-K). Both grafts were well tolerated and osteoconductive. At 2 weeks, there was decreased bone volume and density in the gamma irradiated graft compared to the SCF treated graft, corresponding with a greater inflammatory response histologically and increased Cathepsin-K expression. Catabolic activity predominated at 4 weeks, with both grafts undergoing significant resorption and remodeling inside the defect. Alkaline Phosphatase expression was greater in the SCF group at both time points indicative of a more anabolic response. Allograft bone sterilized with either gamma irradiation or SCF treatment was osteoconductive and capable of healing a critical sized tibial defect in a rabbit. Gamma irradiated allografts elicited an acute inflammatory reaction when implanted which may increase the amount of graft resorption compared to the SCF treated bone.     

Alterations of human acellular tissue matrix by gamma irradiation: histology, biomechanical property, stability, in vitro cell repopulation, and remodeling

AlloDerm, a processed acellular human tissue matrix, is used in a number of surgical applications for tissue repair and regeneration. In the present work, AlloDerm serves as a model system for studying gamma radiation-induced changes in tissue structure and stability as well as the effect of such changes on the cell-matrix interactions, including cell repopulation and matrix remodeling. AlloDerm tissue matrix was treated with 2-30 kGy gamma irradiation at room temperature. Gamma irradiation reduced the swelling of tissue matrix upon rehydration and caused significant structural modifications, including collagen condensation and hole formation in collagen fibres. The tensile strength of AlloDerm increased at low gamma dose but decreased with increasing gamma dosage. The elasticity of irradiated AlloDerm was reduced significantly. Calorimetric study showed that gamma irradiation destabilized the tissue matrix, resulting in greater susceptibility to proteolytic enzyme degradation. Although gamma irradiation did not affect in vitro proliferation of fibroblast cells, it promoted tissue degradation upon cell repopulation and influenced synthesis and deposition of new collagen.     

Calorimetric study of extracellular tissue matrix degradation and instability after gamma irradiation

Native extracellular tissue matrix (ECM) is increasingly used for tissue repair and regeneration. The kinetics of gamma irradiation damage on human dermis ECM was studied by differential scanning calorimetry (DSC). Dermis ECM was irradiated at a low-dose rate of 0.23 kGy h(-1) in order to study the progression of ECM damage as the gamma dose increased from 0 to 32 kGy. The study showed that the effect of gamma irradiation above 2 kGy was predominantly peptide chain scission. As the gamma dose increased, the stability of irradiated ECM decreased further, and multiple ECM domains of different stability were detected. Even a moderate gamma dose (7-12 kGy) could decrease the onset denaturation temperature of ECM to below body temperature. DSC analysis also showed partial and spontaneous protein denaturation in gamma-irradiated, rehydrated ECM at 37 degrees C. In vitro rehydration tests confirmed that a significant fraction of the irradiated ECM disintegrated into minute ECM fragments at 37 degrees C, although the irradiated ECM appeared to be normal at 4 degrees C and room temperature. DSC data were correlated well to effects of gamma irradiation on ECM microstructure, mechanical property and in vitro cell response reported earlier by us. A model was presented to describe the kinetics of gamma-irradiation-induced alterations of tissue ECM properties.     

Irradiated Cerastes cerastes venom as a novel tool for immunotherapy

Immunotherapy is the most effective treatment for the snake bites. The antivenoms are commonly obtained by hyperimmunization of animals that suffer from venom toxicity. The present report describes gamma irradiation effects on Cerastes cerastes venom. Doses of 1 kGy and 2 kGy gamma radiations were used for venom detoxification. These treated venoms did not have any residual lethal effects until 10 LD₅₀. Immunological analysis of sera raised against native and irradiated venoms, showed that elicited antibodies to irradiated venoms were able to recognize native venom. Anti-2 kGy irradiated venom had more protective ability than anti-native venom, as tested in mice.     

Irradiated Cerastes cerastes Venom as a Novel Tool for Immunotherapy

Immunotherapy is the most effective treatment for the snake bites. The antivenoms are commonly obtained by hyperimmunization of animals that suffer from venom toxicity. The present report describes gamma irradiation effects on Cerastes cerastes venom. Doses of 1 kGy and 2 kGy gamma radiations were used for venom detoxification. These treated venoms did not have any residual lethal effects until 10 LD₅₀. Immunological analysis of sera raised against native and irradiated venoms, showed that elicited antibodies to irradiated venoms were able to recognize native venom. Anti-2 kGy irradiated venom had more protective ability than anti-native venom, as tested in mice.     

Comparison of two different methods for inactivation of viruses in serum.

In order to compare protocols for inactivation of viruses potentially present in biological specimens, three different model viruses were treated in bovine serum by two different inactivation methods: samples were subjected either to chemical inactivation with ethylenimine (El) at concentrations of 5 and 10 mM at 37 degrees C for periods up to 72 h or to electron-beam irradiation in frozen and liquid form with doses varying between 11 and 46 kGy. The chemical inactivation resulted in nonlinear tailing curves in a semilogarithmic plot of virus titer versus inactivation time showing non-first-order kinetics with respect to virus titer. The time for inactivation of 7 log10 units of porcine parvovirus (PPV) was about 24 h for both El concentrations, whereas 5 log10 units of bovine viral diarrhea virus (BVDV) was inactivated in 2 h for both El concentrations and 6 log10 units of porcine enterovirus (PEV) was inactivated within 3 h. The inactivation with electron-beam irradiation resulted in almost linear curves in a semilogarithmic plot of virus titer versus irradiation dose, reflecting a first-order inactivation. The rate of inactivation was almost twice as fast in the liquid samples compared to the rate in frozen ones, giving values of the doses needed to reduce virus infectivity 1 log10 unit for inactivation of PPV of 11.8 and 7.7 kGy for frozen and liquid samples, respectively, whereas the corresponding values for BVDV were 4.9 and 2.5 kGy, respectively, and those for PEV were 6.4 and 4.4 kGy, respectively. The nonlinear inactivation with El makes it impossible to extrapolate the curves beyond the virus detection limit and thereby predict the necessary time for complete inactivation, i.e., to a level beyond the detection limit, of virus in a given sample. The first-order inactivation obtained with electron-beam irradiation makes such a prediction possible and justifiable. The two methods are discussed with respect to their different kinetics and applicability under different circumstances and criteria for inactivation, and considerations for choice of method are discussed.     

The effects of high dose irradiation on the cross-linking of vitamin E-blended ultrahigh molecular weight polyethylene

Vitamin E-stabilized, highly cross-linked ultrahigh molecular weight polyethylene (UHMWPE) is a promising oxidation and wear resistant UHMWPE with improved mechanical strength in comparison with the first generation, irradiated and melted UHMWPE. One approach of incorporating vitamin E in UHMWPE is through blending of vitamin E in UHMWPE powder followed by consolidation and radiation cross-linking. However, radiation cross-linking efficiency of UHMWPE decreases in the presence of vitamin E. Therefore an optimum vitamin E concentration and radiation dose level need to be determined to achieve a cross-link density comparable to 100-kGy irradiated and melted UHMWPE, which has shown excellent wear properties in vivo. We investigated the cross-link density and mechanical properties of vitamin E-blended UHMWPEs as a function of vitamin E concentration in the blend and gamma irradiation doses up to 200kGy. We found that 0.3wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 65kGy for virgin UHMWPE and 1.0wt% vitamin E-blended UHMWPE could not be cross-linked above a cross-link density achieved at a radiation dose of 25kGy for virgin UHMWPE even when the these UHMWPEs were irradiated to a radiation dose of 200kGy. In addition, higher plasticity at vitamin E concentrations at and above 0.3wt% indicated that increased chain scissioning may be prevalent. Since the wear resistance of this irradiated UHMWPE would be expected to be low, vitamin E concentrations equal to or above 0.3wt% are not recommended for subsequent irradiation to achieve a wear resistant cross-linked UHMWPE. The long-term oxidative stability of irradiated blends with low vitamin E concentrations has yet to be studied to determine an optimum between cross-link density and long-term oxidative stability.     

Activity of antibodies against Salmonella dublin, Toxoplasma gondii, or Actinobacillus pleuropneumoniae in sera after treatment with electron beam irradiation or binary ethylenimine.

Viral contamination of biological material may constitute a risk when samples are exchanged between countries, and it may be necessary to subject the material to an inactivation treatment. The present study investigated possible adverse effects on antibody activity subsequent to either electron beam irradiation or binary ethylenimine (BEI) treatment. The treatments were performed with sera obtained from pigs or cattle. For each treatment level, the posttreatment activity was plotted against the pretreatment activity, and regression analyses were carried out. The slope of the regression line was used as an estimate for the relative posttreatment activity. For a Toxoplasma gondii indirect enzyme-linked immunosorbent assay (ELISA) and agglutination assay as well as for a Salmonella dublin indirect ELISA, the posttreatment activity was more than 89% of the pretreatment activity when the samples were irradiated in the frozen state (on dry ice) with up to 46. kGy or when they were treated with 5 or 10 mM BEI for up to 48 h. The samples were more sensitive to irradiation in the liquid state. Thus, samples irradiated with 22.6 kGy retained 98% of their activity in the indirect ELISA when they were irradiated in the frozen state on dry ice but only 35% of their activity when they were irradiated in the liquid state at 0 degrees C. The patterns seen in an S. dublin blocking ELISA and an Actinobacillus pleuropneumoniae complement fixation assay differed in that samples with a low level of pretreatment activity were subject to a relatively greater decrease in activity than samples with a high level of pretreatment activity. The complement fixation assay was particularly sensitive to irradiation of serum. It is concluded that serum samples retain sufficient activity by both methods of virus inactivation, especially when used in indirect ELISA or in the T. gondii agglutination assay.     

Crosslinking of poly(L-lactide) nanofibers with triallyl isocyanutrate by gamma-irradiation for tissue engineering application

The radiation crosslinked poly(L-lactide) (PLLA) electrospun nanofibers have been developed with improved thermal stability and mechanical properties. Trially isocyanurate (TAIC) were added into PLLA solution at different weight ratios (1, 3, and 5%) and electrospun into nanofibrous mats, the mats were then irradiated by gamma ray at different radiation doses (5, 10, and 25 kGy) to crosslink the PLLA chains. Their surface morphology, thermal properties, mechanical properties, and biodegradation properties were investigated and compared before and after gamma irradiation. Furthermore, the in vitro biocompatibilities were also evaluated by using mouse L929 fibroblasts. The results indicated that the efficient crosslinking networks can be generated when the TAIC content is higher than 3%. The thermal stability and tensile mechanical properties were significantly increased at higher irradiation dose of 10 and 25 kGy. However, radiation dose at 25 kGy have an adverse effect on the thermal stability of crosslinked samples due to thermal degradation induced by irradiation, the crosslinked samples irradiated at 10 kGy exhibited the best enzymatic degradation. The in vitro results also revealed that the crosslinked PLLA/TAIC composite nanofibers did not induce cytotoxic effects and are suitable for cell growth. Therefore, the crosslinked PLLA nanofibers are one of the promising materials for future tissue engineering applications.     

Structure modification of UHMWPE used for total joint replacements

Modification of ultrahigh-molecular-weight polyethylene (UHMWPE) consisting of a combination of gamma irradiation and subsequent thermal treatment has been performed in order to investigate the resultant changes to its supramolecular structure. In the first step the polymer was irradiated by gamma rays at laboratory temperature under nitrogen. Five radiation doses (25, 50, 100, 150, and 200 kGy) were applied at two dose rates (0.25 and 2.5 kGy/h). In the second step the irradiated samples were thermally treated above the UHMWPE melting temperature. Insoluble fraction, crystallinity (fraction), and lamellar periodicity were determined as functions of dose and dose rate for irradiated samples before and after thermal treatment. Both modification steps were shown to produce substantial changes in the UHMWPE structure.     

Apoptosis induced by gamma irradiation of<Emphasis Type="Italic"> Taenia solium</Emphasis> metacestodes

Gamma irradiation of food is considered a possible approach to control food-borne diseases. In cysticercosis, previous studies have shown that irradiating (with 0.3 kGy) pork infected with Taenia solium larvae completely inhibits growth of the parasite. This study was conducted to evaluate the mechanisms that induce the effect of gamma irradiation on metacestodes of T. solium. Metacestodes were obtained from several infected pigs and irradiated with a dose of 0.3 kGy. The viability of the metacestodes was evaluated by their capacity to evaginate in vitro and in vivo development to tapeworms after they were orally infected into prednisolone-treated golden hamsters. Using the typical ladder pattern of fragmented DNA and the TdT-mediated DUTP-nick-end labeling assay, apoptosis was evaluated in metacestodes after irradiation and in the scolices and tapeworms recovered from infected hamsters at 21 days post-infection. Apoptosis was observed in the structure of scolices obtained from hamsters at 21 days post-infection with irradiated metacestodes, This study provides evidence of the existence of apoptosis in the irradiated metacestodes of T. solium and helps elucidate the possible mechanisms that are involved when gamma irradiation inhibits the normal development of the T. solium metacestode into the adult worm.     

Identification and quantification of irradiation in UHMWPE through trans-vinylene yield

trans-Vinylene unsaturations generated in ultrahigh-molecular-weight polyethylene (UHMWPE) after radiation treatment scales linearly with the absorbed dose level, and can serve as an internal dosimeter for determining the radiation dose level for sterilizing or crosslinking UHMWPE. We measured the trans-vinylene concentration using infrared spectroscopy and generated calibration curves for ultrahigh-molecular-weight polyethylene (ram-extruded GUR 1050) after cold e-beam, cold gamma, and warm e-beam irradiations. The trans-vinylene content increased linearly with the absorbed dose level in all cases except with cold gamma irradiation at dose levels higher than approximately 70 kGy. At that dose level the trans-vinylene content of the warm irradiated samples was higher than those of the cold irradiated samples. Following postirradiation melting, the trans-vinylene content of the gamma irradiated samples increased. Such trans-vinylene quantification can be an internal dosimeter for crosslinked UHMWPE when radiation is used for improving its wear resistance or sterilizing the device made thereof. It can also be utilized to assess the spatial uniformity of the absorbed radiation dose level.     

Flexural strength of experimentally filled resins made of electron beam irradiated silica fillers

This study investigated the influence of different silica fillers on the flexural strength of experimentally filled resins. Hydrophilic (non-silanated) silica, hydrophobic silica modified by organofunctional silane, and silica modified by organofunctional silane that additionally contains polymerizable carbon double bonds were assigned into further subgroups: the first subgroup was electron beam irradiated with 10 kGy (dose rate) and the second with 30 kGy, whereas the third constituted the non-irradiated control group. In total, nine experimentally filled resin blends were mixed. Rectangular specimens were constructed, and a flexural strength test was performed. Regardless of the type of silica, specimens constructed of blends containing non-irradiated fillers showed the lowest flexural strength in comparison to their corresponding irradiated groups. With increasing dose rates from 10 to 30 kGy, filler irradiation prior to blend mixing resulted in slightly increased flexural strength values for hydrophilic as well as for organofunctional silanated silica. Specimens constructed of blends with fillers that were not only modified by silane containing polymerizable carbon double bonds but were additionally irradiated showed the highest flexural strength. The results of this study indicate that the flexural strength of filled resins could be enhanced by advance preparation of silica fillers with silane coupling agents followed by electron beam irradiation.     

Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation.

Because of the cumbersome conditions experienced in a maximum containment laboratory, methods for inactivating highly pathogenic viruses were investigated. The infectivity of Lassa, Marburg, and Ebola viruses was inactivated without altering the immunological activity after radiation with Co60 gamma rays. At 4 degrees C, Lassa virus was the most difficult to inactivate with a rate of 5.3 X 10(-6) log 50% tissue culture infective dose per rad of CO60 radiation, as compared with 6.8 X 10(-6) log 50% tissue culture infective dose per rad for Ebola virus and 8.4 X 10(-6) log 50% tissue culture infective dose per rad for Marburg virus. Experimental inactivation curves, as well as curves giving the total radiation needed to inactivate a given concentration of any of the three viruses, are presented. We found this method of inactivation to be superior to UV light or beta-propiolactone inactivation and now routinely use it for preparation of material for protein-chemistry studies or for preparation of immunological reagents.     

Trace concentrations of vitamin E protect radiation crosslinked UHMWPE from oxidative degradation

The effect of very low concentrations of Vitamin E on the stability and mechanical behavior of UHMWPE remains unknown. We tested the hypothesis that the oxidation resistance of Vitamin E-blended UHMWPE would be influenced by trace doses of antioxidant, resin, and radiation treatment. Trace concentrations (< or =500 ppm w/w%) of alpha-tocopherol (Vitamin E) were blended separately with GUR 1020 and 1050 resins and molded into disks. From each disk, three groups of 10 mm thick blocks were machined: (1) no irradiation (control); (2) 30 kGy of gamma irradiation in nitrogen; and (3) 75 kGy of gamma irradiation in air. Specimens were subjected to three aging protocols: (a) no aging (control); (b) two weeks and (c) four weeks of accelerated aging in accordance with ASTM F 2003 (i.e., 70 degrees C and 5 atm oxygen). The minimum concentration of Vitamin E needed to stabilize UHMWPE during our accelerated tests depended upon the method of radiation processing. For the 30 and 75 kGy irradiated materials, the addition of 125 ppm or more Vitamin E was sufficient to maintain baseline mechanical and chemical properties through two weeks of accelerated aging. For these groups, the addition of 375 ppm or 500 ppm, respectively, was necessary to maintain baseline mechanical and chemical properties throughout the four-week accelerated aging period. UHMWPE resin molecular weight did not have an effect on oxidation behavior. The results of this experiment therefore supported our hypotheses that trace concentrations of Vitamin E, coupled with radiation treatment-but not resin grade-influence the mechanical and oxidative degradation behavior of UHMWPE.     

Characterization of the effects of x-ray irradiation on the hierarchical structure and mechanical properties of human cortical bone

Bone comprises a complex structure of primarily collagen, hydroxyapatite and water, where each hierarchical structural level contributes to its strength, ductility and toughness. These properties, however, are degraded by irradiation, arising from medical therapy or bone-allograft sterilization. We provide here a mechanistic framework for how irradiation affects the nature and properties of human cortical bone over a range of characteristic (nano to macro) length-scales, following x-ray exposures up to 630 kGy. Macroscopically, bone strength, ductility and fracture resistance are seen to be progressively degraded with increasing irradiation levels. At the micron-scale, fracture properties, evaluated using insitu scanning electron microscopy and synchrotron x-ray computed micro-tomography, provide mechanistic information on how cracks interact with the bone-matrix structure. At sub-micron scales, strength properties are evaluated with insitu tensile tests in the synchrotron using small-/wide-angle x-ray scattering/diffraction, where strains are simultaneously measured in the macroscopic tissue, collagen fibrils and mineral. Compared to healthy bone, results show that the fibrillar strain is decreased by ～40% following 70 kGy exposures, consistent with significant stiffening and degradation of the collagen. We attribute the irradiation-induced deterioration in mechanical properties to mechanisms at multiple length-scales, including changes in crack paths at micron-scales, loss of plasticity from suppressed fibrillar sliding at sub-micron scales, and the loss and damage of collagen at the nano-scales, the latter being assessed using Raman and Fourier Transform Infrared spectroscopy and a fluorometric assay.     

Endothelial cell attachment to the gamma irradiated small diameter polyurethane vascular grafts

Previously we have fabricated the small diameter polyurethane (Pellethane 2363-80A, abbreviated PU) vascular grafts that were modified by epoxy-crosslinked gelatin (abbreviated gelatin) and an RGD-containing protein (abbreviated CBD-RGD) to facilitate the endothelial cell (EC) seeding on the surface. In this study, the biocompatibility of such surface after freeze-drying and gamma irradiation was evaluated. The contact angle of the irradiated PU dropped a little and the ESCA spectra revealed oxygen bonding. The increases in the amount of extractables as well as in the molecular weight distribution were observed. The mechanical properties decreased only slightly. The irradiated PU surface showed enhanced EC affinity that persisted after several months of storage. Gelatin, CBD-RGD (used with either gelatin or PU), and PU modified by gelatin and CBD-RGD all demonstrated higher EC affinity after freeze-drying and gamma irradiation (2.5 Mrad). The positive cellular effect remained after storage. Based on these results, freeze-drying followed by gamma irradiation at 2.5 Mrad is a proper way to process and store these vascular grafts.     

Mechanical properties of gamma irradiated morselized bone during compaction

This study examined the effects of gamma irradiation on the compressive properties of morselized cancellous bone from human femoral heads. Twelve bone samples, mean age of 68 years (range 92-39), were divided into 3 groups (N=12) of varying irradiation level (0, 15 and 25 kGy). Each specimen was compacted in a controlled fashion in steps of 0.5 mm at 0.5 mm/min (up 12 mm). The load and stiffness increased with compaction, but this relationship was not linear. There was no statistical significant difference in the compacting load or stiffness between groups (p>0.05) until the last 1 mm of compaction, where the 25 kGy group were significantly stiffer compared to controls but not different to the 15 kGy group. This may be due to decreased interlocking of bone particles caused by higher irradiation levels resulting in a stiffer graft.     

Influence of the remelting process on the fatigue behavior of electron beam irradiated UHMWPE

Electron beam irradiation at doses below 150 kGy is a widely used technique to obtain highly crosslinked ultra-high-molecular-weight polyethylene (UHMWPE). Its current use in total joint replacement components may improve wear resistance and decrease UHMWPE particle debris. However, currently used post-irradiation thermal treatments, which aim to decrease the free radicals within the material, introduce microstructural changes that affect UHMWPE mechanical properties, particularly the fatigue strength. This influence may be crucial in total knee replacements, where fatigue-related damage limits the lifespan of the prosthesis. Therefore, more studies are required to understand UHMWPE fatigue after current crosslinking protocols. This study was planned to evaluate the influence of UHMWPE remelting after irradiation on the material fatigue resistance. The remelting was achieved at 150 degrees C for 2 h on UHMWPE previously irradiated at 50, 100, and 150 kGy. Fatigue evaluation included short-term tests under cyclic tensile stress with zero load ratio, R = 0, and 1 Hz. In addition, stress-life testing was performed using 12% yield as the criterion for failure. Near-threshold fatigue crack propagation experiments were also performed at a frequency of 5 Hz, and crack length was measured in nonthermally treated and remelted irradiated UHMWPE. Crystallinity percentage was calculated from DSC measurements. The results pointed out that irradiation positively contributed to total life analysis, but the further remelting process decreased the flaw initiation resistance. On the other hand, both processes negatively affected the fatigue resistance of notched components. From a clinical point of view, the results suggest that the material fatigue behavior should be carefully studied in new UHMWPE to avoid changes related to material processing.