Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue

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Journal of Orthopaedic Research

Journal of Orthopaedic Research 23 (2005) 838-845

www.elsevier.com/locate/orthres

Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue I2 Ozan Akkus *, Ryan M. Belaney, Prasenjit Das Department of Bioengineering, The University of Toledo, 5035 Nitschke Hall, 2801 W. Bancroft Street, Mail Stop 303, Toledo, OH 43606 3390, United States Accepted 25 January 2005

Abstract

Terminal sterilization of bone allografts by gamma radiation is often essential prior to their clinical use to minimize the risk of infection and disease transmission. While gamma radiation has efficacy superior to other sterilization methods it also impairs the material properties of bone allografts, which may result in premature clinical failure of the allograft. The mechanisms by which gamma radiation sterilization damages bone tissue are not well known although there is evidence that the damage is induced via free radical attack on the collagen. In the light of the existing literature, it was hypothesized that gamma radiation induced biochemical damage to bone’s collagen that can be reduced by scavenging for the free radicals generated during the ionizing radiation. It was also hypothesized that this lessening of the extent of biochemical degradation of collagen will be accompanied by alleviation in the extent of biomechanical impairment secondary to gamma radiation sterilization. Standardized tensile test specimens machined from human femoral cortical bone and specimens were assigned to four treatment groups: control, scavenger treated-control, irradiated and scavenger treated-irradiated. Thiourea was selected as the free radical scavenger and it was applied in aqueous form at the concentration of 1.5 M . Monotonic and cyclic mechanical tests were conducted to evaluate the mechanical performance of the treatment groups and the biochemical integrity of collagen molecules were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native mechanical properties of bone tissue did not change by thiourea treatment only. The effect of thiourea treatment on mechanical properties of irradiated specimens were such that the post-yield energy, the fracture energy and the fatigue life of thiourea treated-irradiated treatment group were 1.9-fold, 3.3-fold and 4.7-fold greater than those of the irradiated treatment group, respectively. However, the mechanical function of thiourea treated and irradiated specimens was not to the level of unirradiated controls. The damage occurred through the cleavage of the collagen backbone as revealed by SDS PAGE analysis. Irradiated specimens did not exhibit a noteworthy amount of intact a-chains whereas those irradiated in the presence of thiourea demonstrated intact a-chains. Results demonstrated that free radical damage is an important pathway of damage, caused by cleaving the collagen backbone. Blocking the activity of free radicals using the scavenger thiourea reduces the extent of damage to collagen, helping to maintain the mechanical strength of sterilized tissue. Therefore, free radical scavenger thiourea has the potential to improve the functional life-time of the allograft component following transplantation. 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Bone graft; Gamma radiation; Sterilization; Cortical bone; Collagen; SEM; Free radicals; Free radical scavengers; Thiourea; Biomechanical

* The process described herein which describes reducing the biomechanical and biochemical impairment of gamma radiation sterilized graft components is patent pending. * Corresponding author. Tel.: +1419 530 8256; fax: + I 419 530 8076. E-mail address: [email protected] (0.Akkus).

Introduction Each year, an estimated 450,000 allografts are transplanted in the US for repair of fractures and damage

0736-0266/$ - see front matter 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi:lO. 1016/j.orthres.2005.01.007

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caused by illness and injury, replacing bone lost in tumor removal, and reconstruction of skeletal defects. Allograft popularity is related both to its biocompatibility and to its suitability for anatomical matching to repair defects. Regardless of their expediency, risk of infection and disease transmission through allografts is a concern and terminal sterilization is often essential [26]. Gamma radiation has been widely used for sterilization of bone allografts due to its efficacy against viral and bacterial disease transmission [5,6,8]. However, gamma radiation sterilization impairs the material properties of bone [1,2,5,12,18,39]which is a major clinical concern since bone grafts are used in load bearing applications [131. In assessing the mechanical properties of bone irradiated with gamma radiation, it was commonly observed that pre-yield (elastic) behavior of cortical bone tissue is unaffected while post-yield (plastic) properties suffer a significant reduction as a result of radiation sterilization [1,2,5,12,18,39]. Degradation in post-yield properties results in the loss of tissue ductility and gamma radiation sterilized bone tissue becomes brittle [ 1,2,5, 12,18,39]. This behavior is hypothetically explained by the dependency of pre-yield properties of cortical bone tissue on the mineral phase and the post-yield properties on the collagen [7,43]. Therefore, it is believed that the collagen phase is more vulnerable to gamma radiation than the mineral phase of cortical bone tissue. Besides gamma radiation induced embrittlement of bone, age [32,45] and disease [20,40] related alterations in collagen biochemistry have also been documented to cause bone brittleness. Thus, it is widely accepted that the integrity of bone’s collagen profoundly effects the mechanical strength and fracture resistance of bone tissue [41]. The effect of gamma radiation on the biochemistry of collagen has also been extensively investigated for collagen extracted from tendons [3,9,34]. It is shown that gamma radiation leads to scission of the peptide backbone [3,9], and reduces the concentration of intermolecular cross-links in tendon collagen [34]. Similarly to tendon, destruction of the peptide backbone of bone’s collagen [171 and reduction in intermolecular cross-link density [101 have been reported for human femoral cortical bone secondary to gamma radiation sterilization. The majority of gamma radiation damage is induced by damaging species (i.e. free radicals) resulting from the radiolysis of water molecules [16,37]. In this regard, collagen is a primary target for radiation-based free radical attack due to the significant amount of water bound to its structure [36]. Free radicals resulting from the radiolysis of water react with target molecules within a lifetime on the order of 0.01-1 ns and render irrecoverable changes in the target molecule’s chemical structure [ 161. Particularly, the hydroxyl (OH’) free radical induces the greater portion of the in vivo and in vitro damage to biological systems during gamma radiation [19,37,42].

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Supporting the existence of a water-based free radical attack, bone irradiated at -78 “C was less brittle and had less collagen damage than when irradiated at room temperature [17]. So as to explain this observation, Hamer et al. speculated that the mobility of water molecules is restrained in the frozen state and, in turn, fewer free radicals are generated [17]. It seems likely that gamma radiation impairs the structure of the collagen matrix by water-based free radical attack [lo, 171. However, data supporting this assertion are scant. The purpose of the current study was to minimize the mechanical impairment of gamma radiation sterilized bone tissue by limiting the free radical damage pathway to the collagen phase. The following experiments tested the hypothesis that human cortical bone tissue sterilized in the presence of the free radical scavenger thiourea will be mechanically superior (i.e. less brittle) to cortical bone tissue sterilized in the absence of thiourea. For this purpose specimens were subjected to monotonic and cyclic tensile tests to assess the potential of the free radical scavenger thiourea for reducing gamma radiation related impairment of bone. The radioprotective effect of thiourea on collagen was investigated qualitatively by SDS-PAGE analysis and by fractographic investigation of failure surfaces via SEM.

Materials and methods Prepurution of tensile test specimens

Femurs from three male cadavers (ages 31, 31, 38) were obtained from the Musculoskeletal Transplant Foundation (Jessup, PA, USA). The diaphyses were sectioned into two segments, each approximately 55 mm long, using a hacksaw. The first segment was taken immediately distal to the minor trochanter and the second distally from the first segment. A low-speed metallurgical saw with a diamond coated blade (SouthBay Tech, CA, USA) was used to cut the rings into the four anatomical quadrants. One millimeter thick wafers were sectioned from the quadrants within the circumferential-longitudinal plane (parallel to osteonal orientation) using the low-speed metallurgical saw. Wafers were then cut into beams with final dimensions of 40 mm x 5 mm x 1 mm. Coupon shaped tensile test specimens were machined from the beams by reducing the width of the mid-gage region using a table-top milling machine (Sherline, CA, USA) and a 0.5” diameter end-mill. The gage region had the final dimensions of 2 mm x 1 mm x 16 mm. Specimens were kept wet with calcium supplemented saline solution by using an air-pressure driven nozzle spray during the milling process. Four treatment groups were included in the study: controls (C), thiourea treated controls (1.5C), irradiated (IR) and thiourea treated-irradiated (1.5IR). Specimens were randomly assigned to each of the four treatment groups. Fifteen monotonic and four fatigue specimens were assigned to each treatment group, reaching a total of 76 specimens. Specimens were kept wet at all times and stored at -40 “C. Thiourea treatment and sterilization

Free radical scavengers are substances that inhibit free radical damage to a target molecule by direct chemical reaction with the radical or by minimizing the formation of the radical. Free radical damage to collagen extracted from tendons was previously inhibited by using the scavengers thiourea [3,25] and cysteamine [25]. Riedle and Kerjaschki

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have demonstrated a similar protective effect of the antioxidant enzyme, catalase, against free radical damage to type IV collagen [33]. In this study thiourea (CH4N2S, 76.12 Da) was selected for scavenging of free radicals for several reasons. The supramolecular level porosity of bone tissue allows for the penetration of molecules with a molecular weight less than 300 Da [38]. The lack of charged groups in thiourea further facilitates its diffusion in bone. Thiourea has low toxicity and its compounds are used for the treatment of various diseases [23,29]; however, its carcinogenicity is questionable [14,31]. Specimens which received thiourea treatment (1.5C and 1.5IR) were placed in polyethylene containers in groups of 20 and soaked in 40ml of 1.5 M thiourea solution supplemented with calcium [IS] and protease inhibitors to minimize leaching of mineral and bacterial degradation. This concentration was determined based on pilot tests in which tensile specimens machined from bovine bone were treated at concentrations of 0.1 M, 0.5 M and 1.5 M. Since none of these concentrations altered the native mechanical properties of bovine bone the maximum concentration of 1.5 M was selected. In addition, the solubility of thiourea in aqueous environment is such that it was not possible to obtain concentrations greater than 1.5 M. Solutions were replaced every 3 days and the entire treatment lasted for 14 days. The treatment was carried out at 4 “C to minimize bacterial degradation. The control and irradiated treatment groups were kept under similar conditions except thiourea was absent from the solutions in which they are maintained. Specimens were individually wrapped in gauze pads dipped in calcium supplemented saline solution and placed in polyethylene bags. Treatment groups which received gamma radiation were placed in polystyrene coolers filled with dry ice and mailed to Steris Corporation (Steris Corporation, Columbus, OH, USA) for gamma radiation sterilization. Control samples were placed in the same type of polystyrene coolers filled with dry ice and placed on the lab bench until the irradiated specimens were sent back to our facilities. Samples were irradiated at an average dose of 36.4 kGy as measured by perspex dosimeters on the site of irradiation. The standard dose range for sterilization of bone grafts is 25 kGy to 35 kGy; thus, the level of radiation in this study was slightly greater than this standard range. Monotonic tensile tests

The specimen was locked into the grips of an electromagnetic mechanical testing machine (ELF 3200, Enduratec, Minnetonka, MN, USA) and the clip-on strain gage extensometer (Epsilon, Jackson, WY, USA) was attached to the gage region using orthodontic heavy gauge rubber bands (3M, 3/16” Heavy, MN, USA). Monotonic tests were performed under strain control at the rate of 1.5%/s. The load was measured by a 400 N load cell (Honeywell-Sensotec, Columbus, OH, USA). Load-strain data were acquired at the rate of 400 Hz. Tensile specimens were kept wet by drips of calcium supplemented saline solution at ambient temperature. Stress was calculated by dividing the measured load with the cross-sectional area at the reduced gage region. Stresdstrain curves were constructed from the raw data and mechanical properties were calculated using custom-written software (Matlab, The Mathworks, Natick, MA, USA). The yield point (limit for elastic deformability) was determined by the 0.2% offset-method. The elastic deformation capacity (elastic energy) was calculated from the area within the elastic region whose endpoint was defined by the yield limit. The energy required to fracture the specimen (fracture energy) was calculated from the area under the entire stress-strain curve. The post-yield deformation capacity (post-yield energy) was calculated as the difference between the fracture energy and the elastic energy. Tensile fatigue life

Fatigue tests were conducted under load-control using sinusoidal waveform at 2 Hz. The initial load level was arranged such that the resulting strain acting on the specimen was 0.2% which corresponded to about 25% of the average yield strain obtained from monotonic tests of control specimens. This level of strain is less than the reported threshold strain of 0.25% above which cortical bone fails at a much faster rate under tensile fatigue. Therefore, the fatigue loading was within the high-cycle range and the initial strain was physiologically relevant. The minimum load was set at 10% of the maximum load

value. Specimens were kept wet by continuous drip of calcium supplemented saline solution at ambient temperature during the entire loading. If the test specimen did not break by 300,000 cycles ( x 2 days) the test was terminated and the failure cycle was recorded as 300,000. Scanning electron microscopy

Two monotonic and two fatigue specimens were selected from each treatment group and their fracture surfaces were sputter coated with gold and qualitatively investigated via scanning electron microscopy (JEOL, Akishima, Japan). SDS-PAGE analysis of collagen cc-chains For each treatment group, the grip regions of four randomly picked monotonic tensile specimens were cut-off and pooled. Tissue was frozen in liquid nitrogen and pulverized manually using a stainless-steel mortar and pestle. The powder was defatted and dehydrated in ethanol for 30 min, lyophilized overnight (Labconco, Kansas City, MO, USA) and demineralized in 0.5 M EDTA adjusted to pH 7.2 for 3 days, at 4°C. The EDTA solution was centrifuged and the precipitate was placed in distilled water and dialyzed (1000 Da MW cut-off) against ultrapure distilled water for 3 days to clear the mineral ions from the solution. Dialyzed samples were centrifuged and the precipitates were solubilized in 0.5 M acetic acid solution with pepsin (1O:l weight ratio of bone/enzyme) for 48 h at 4 “C. Solubilized collagen in the supernatant was precipitated by addition of NaCl to attain a concentration of 2.0 M for 24 h and the precipitates were recovered by centrifuging at 35,OOOg for 1 h. The pellet was redissolved in 0.5 M acetic acid, dialyzed against 0.2 M acetic acid solution and lyophilized to obtain soluble collagen. Salt precipitated collagen and collagen standards (Sigma-Aldrich, St. Louis, MO, USA) were run on yo5 SDS-PAGE slabs [21] at a concentration of 5 mg/ml, the gel was stained with Coomassie blue and destained. The locations of bands were verified by molecular weight markers (Sigma-Aldrich, St. Louis MO, USA). Four separate gels were run to confirm the consistency of the resulting gelprofiles. Statistical analyses

Generalized multivariate analysis of variance (MANOVA) was performed to determine the significancesof the main effect of gamma radiation sterilization (the level of significance denoted by ‘ P S T E R ’ ) , the main effect of thiourea treatment (the level of significance denoted and the interaction effects of the two factors (the level of by ‘PTHJO’) on mechanical properties. If MANOsignificance denoted by ‘PsT-TH’) VA of main or interaction effects denoted significance then the difference between any two treatment groups was tested by a MannWhitney U-test (the level of significance denoted by ‘p‘). A difference at the level of p < 0.05 was reported as significant and a difference at the level of 0.05 < p < 0.1 is reported as borderline significant.

Results The elastic, the post-yield and the overall deformation energies of the monotonic specimens suffered significant reductions following gamma radiation sterilization at a dose of 36.4 kGy (Table 1 and Fig. 1). The post-yield and overall deformation energies of bone tissue were dramatically reduced by 70% and 87%, respectively, whereas the elastic energy experienced a relatively modest reduction of 26%. The fatigue life of the irradiated treatment group also suffered a significant reduction of 87% secondary to gamma radiation sterilization. Thiourea treatment alone did not alter the native mechanical properties

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Table 1 Mechanical properties of treatment groups. Values denote mean f standard deviation Thiourea

Marginal meanC 1.5 M

OM **

Resilience [Jlmm'], P s T E R = 0.04, PTHIO= 0.22, PST-TH= 0.36 Gamma radiation (n = 15) Control 0.50 f 0.11 Irradiated 0.43 f 0.08 (26%)'

0.51 f 0.10 0.48 f 0.06a (4%)

Marginal mean'

0.50 f 0.08

0.47 f 0.10

0.51 f 0.10 0.46 f 0.08

*I

Work to fracture [Jlmm3], PSTERc 0.005, PTHIO= 0.47. PST-TH= 0.07 Gamma radiation (n = 15) Control 2.22 f 1.18 Irradiated 0.66 f 0.33 (70%j

1.97 f 1.05 1.23 f 0.54b (44%).

Marginal mean

1.58 f 0.88

1.47 f 1.17

1.97 f 1.05 1.23 f 0.54

I .

Post-yield energy [Jlmm'], PSTER< 0.005. PTHIO= 0.52, PST-TH=0.07 Gamma radiation (n = 15) Control 1.71 f 1.12 Irradiated 0.23 f 0.27 (87%j

0.98 f 1.11

Marginal mean

..

1.46 f 0.98 0.75 f OSlb (56%)' 1.08 f 0.83

Fatigue life [cycleslIOOO], PSTERC 0.005, PTHIO= 0.03, PST-TH=0.03 Gamma radiation (n = 4) Control 3 0 0 fO Irradiated 40 f 31 (87%)'

300+-0 187 +- 86b (38%)'

Marginal mean

225 +- 88

151 ? 141

1.60 f 1.05 0.49 f 0.48

300 f 0 128k 123

**: PSTER, PTHloand PsT-TH designate the level of significance for the main effect of gamma radiation, main effect of thourea treatment and the interaction of main effects, respectively. These values are the outcome of a generalized MANOVA test. a,b: 'a' and 'b' designate a significant differencebetween the groups IR (irradiated) and 1S I R (thiourea-treated-irradiated) at the levels o f p < 0.1 and p < 0.05 respectively, as determined by a Mann-Whitney U-test. c: The marginal mean represents the average value of a given property after pooling the data along a row (i.e. thiourea treatment) or column (i.e. radiation). *: Percent reduction in a given mechanical property with respect to the control group C. The mechanical property value is subtracted from that of the treatment group C and divided by the value of the treatment group C to calculate the percent reduction.

120 -

6

100 -

Thiourea treated Irradiated

h

80-

v

I 60-

e

401/ 20 " ,

0

1

2

3

Strain (%)

Fig. 1. Typical stress-strain curves obtained during the monotonic testing of specimens from different treatment groups. The fracture points are indicated by cross-marks. Curves for thiourea treated control specimens were similar to those of controls.

of bone tissue; none of the variables of thiourea treated controls differed from those of the control specimens (Mann-Whitney U-test p > 0.1) (Table 1). Generalized MANOVA tests revealed that the main effect of thiourea treatment was insignificant for postyield and overall energies; however, the interaction effects demonstrated borderline significance at the level

of PST-TH = 0.07 (Table 1). These observations indicate that the effect of thiourea varies with gamma radiation, i.e., thiourea does not have an effect in the absence of irradiation whereas following irradiation the effect of thiourea on post-yield and fracture energies becomes observable. The effect of thiourea treatment was such that the post-yield and fracture energy values of the treatment group 1.5IR (thiourea treated-irradiated) were 1.9-fold and 3.3-fold greater than those of the treatment group IR (irradiated), respectively (p < 0.05, MannWhitney U-test) (Table 1 and Fig. 1). On the other hand, the post-yield and fracture energy values of the treatment group 1.5IR (thiourea treated-irradiated) were significantly less than those of controls (p < 0.05, Mann-Whitney U-test). Therefore, thiourea treatment had a noteworthy radioprotective effect on the postyield and fracture energies of irradiated specimens; however, the extent of this radioprotective effect was not enough to improve the mechanical properties of sterilized specimens to the level of unirradiated controls. General MANOVA analyses of the elastic energy demonstrated a main effect of gamma radiation only whereas the main effect of thiourea treatment and the interaction effect were insignificant. Therefore, the modest reduction in the elastic deformability of bone

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tissue secondary to gamma radiation sterilization was not reversed by the thiourea treatment. Generalized MANOVA analyses of fatigue life indicated that the main effect of radiation, the main effect of thiourea treatment, and the interaction effect were all significant. The radioprotective effect of thiourea on the fatigue life of gamma radiation sterilized cortical bone was substantial. The fatigue life of the treatment group 1.5IR was 4.7 times greater than the fatigue life obtained from the treatment group IR (p < 0.05, Mann-Whitney U-test). Yet, the mean fatigue life of the treatment group 1SIR was significantly less than

that of the treatment group C , indicating that the fatigue life was not improved to the level of controls. Qualitative inspection of fracture surfaces from monotonic and fatigue specimens suggested different fracture mechanisms between the treatment groups (Fig. 2). Failure surfaces of unirradiated control specimens (C and 1.32) were tortuous at the microscale such that there were lamellar extrusions, indicating the involvement of the microstructure in the fracture process. In contrast, fracture surfaces of irradiated specimens were flat without any meandering of the surface features at the microstructural level, indicating that the

Fig. 2. SEM images of failed osteons typical to each treatment group. The failure pattern of the treatment group 1.5C did not differ from that of controls. Fibrillar extensions on the fracture surfaces are indicated by arrowheads.

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Molecular Control Irradiated 1.5 [MIWeight Irradiated Marker Fig. 3. The effects of gamma radiation and thiourea treatment on the integrity of collagen molecules.

failure occurred at the ultrastructural scale and the final failure propagated without any regard to the microscopic architecture. Fractographic analyses of thiourea treated and irradiated specimens revealed that the failure pattern was qualitatively similar to the tortuous fracture surface of the control specimens as opposed to the featureless fracture surface of irradiated specimens, suggesting that thiourea treatment shifts the failure process from the ultrastructural level back to the microstructural level. SDS-PAGE gel electrophoresis of specimens from C and 1.32 treatment groups demonstrated the al(I), a2(I) (denoting the crl and a2 chains of type I collagen, respectively) and p bands of the collagen molecules (Fig. 3). The presence of these three bands in the lanes of irradiated specimens did not appear to be as strong as unirradiated controls despite similar amounts of solubilized collagen being loaded in all lanes, indicating that collagen molecules were cleaved along their backbones so that the number of intact collagen molecules was greatly diminished. Furthermore, the lanes of irradiated specimens exhibited a smear of stain across their length, indicating that the cleavage of the collagen backbone was extensive. Thiourea treatment helped to alleviate this cleavage to a certain extent as was evident from the reappearance of intact cq 1, a12and p bands in the lanes of irradiated specimens treated with thiourea. The described pattern of behavior was consistent for all of the four aliquots which were run on SDS-PAGE. Discussion

During gamma radiation sterilization highly reactive hydroxyl radicals are formed due to ionization of water

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molecules. These free radicals have been speculated to impair the integrity of collagen molecules [17]. In the light of the existing literature, it was hypothesized that gamma radiation induced biochemical damage to bone’s collagen can be reduced by scavenging for the free radicals generated during the ionizing radiation. It was also hypothesized that this lessening in the extent of biochemical degradation of collagen would be accompanied by reduced biomechanical impairment secondary to gamma radiation sterilization. In support of these hypotheses, qualitative SDS-PAGE analysis demonstrated the presence of intact collagen molecules for those specimens irradiated in the presence of thiourea whereas the collagen molecules of those specimens irradiated without any thiourea treatment were extensively cleaved along their backbone. Maintaining the integrity of collagen molecules resulted in several fold improvement in the mechanical properties of specimens which were sterilized in the presence of thiourea in comparison to those irradiated in the absence of thiourea. The improvement in the mechanical properties of thiourea treated and irradiated specimens was not to the level of unirradiated controls, suggesting that there is a limit for the efficacy of thiourea. It is possible that thiourea cannot perfuse the tissue below a certain supramolecular size scale or cannot scavenge for the free radicals with full efficiency. Also, a portion of gamma radiation induced damage may be caused by mechanisms other than the free radicals. Although the treatment did not salvage the mechanical integrity to the level of controls, the improvement in the fracture resistance of irradiated specimens with thiourea treatment was substantial. It was evident that the improvement in the fracture resistance of irradiated tissue took place through the recovery of post-yield deformability of bone. This observation corroborates the concept that the deformation of the post-yield region of bone tissue is governed by the collagen phase. The relative strengths of fibers, matrix and the interface between them affects the failure pattern of fibrous composite materials profoundly [l 11. If fibers are stronger than the matrix and the interface then the crack travels between the fibers. The fibers bridge the wake of the crack, increasing the resistance to crack propagation. Depending on the strength of the interface and fiber length, these bridging fibers may be pulled out or they rupture prior to failure. This fibrillar bridging also helps to transfer the load at the crack tip to longer distances away from the crack, resulting in an increase in the damage zone size. When the mechanical, biochemical and fractographic observations of this study are taken together, the following hypothesis emerges on the failure of gamma radiation sterilized bone tissue. At the submicroscopic level, mineralized collagen fibrils serve this bridging function [28,44] whereas the matrix surrounding them is composed of extrafibrillar mineral crystals

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[30,35]. Collagen fibrils of control specimens were intact; therefore, the crack growth was likely hindered by crack bridging and the final failure involved the rupture and pull-out of collagen fibrils, as evidenced by the fibrillar extensions on the fracture surfaces of control specimens (Fig. 2). Therefore, the failure was resisted at the supramolecular level and loads were transmitted to the microstructural level as evidenced by the involvement of lamellar extrusions on the SEM images of control specimens. As demonstrated by SDS-PAGE analysis, gamma radiation cleaved the backbone of collagen molecules which rendered the mineralized collagen fibrils amenable to failure at lower loads. Therefore, the crack growth was not countered by the fibrillar bridging and the crack propagated through the fibers indiscriminately. This is evidenced by the dull appearance of the fracture surfaces of irradiated specimens and the lack of involvement of microstructural scale in the failure process. Loads experienced by allografts would be cyclic in nature; therefore, fatigue rather than static loading would be expected to better represent physiological loading. The current study demonstrated a 5-fold increase in the fatigue life of bone tissue subjected to gamma radiation in the presence of the free radical scavenger thiourea. An improvement in the biomechanical performance of irradiated bone tissue has important clinical repercussions from the perspective of increased functional life-time of the allograft component following transplantation. Long-term survival of allografts may provide additional valuable time for the host structures to recover their strength. Consequently, clinical complications related to failure of allografts may be prevented. The termination of fatigue loading at a pre-determined number of cycles (300,000) nullified the standard deviation of control specimens in an artificial manner, potentially creating a ‘false significant’difference between controls and irradiated specimens. Fatigue tests were performed under constant load which generated an initial strain level of 0.2%. At this initial strain level, control specimens would be expected to fail by about 4,000,000 loading cycles [24,27]. Therefore, had the control specimens been loaded until failure the resulting mean fatigue life would be high enough to ensure a significant difference between controls and irradiated treatment groups. The results of this study have important implications for bone banking and bone graft technology as well. The use of free radical scavengers would allow for biomechanically more stable grafts and the application of higher doses. The most important limitation of free radical scavengers is that they may have radioprotective effect on viral and bacterial DNA; thus, sterility assurance may be affected negatively [4,22]. Basha et al. have reported that glycerol had a radioprotective effect on Salmonella whereas cysteamine provided minimal protection [4]. Therefore, the protective effect of a scavenger on pathogens needs to be assessed prior to its utilization

towards the protection of collagen matrix. Additional investigations need to be carried out to identify scavengers which protect the biochemical and biomechanical integrity of connective tissue while maintaining the sterility. Acknowledgments

This study was made possible through funding from the Musculoskeletal Transplant Foundation (MTF). The authors would like to further acknowledge the MTF for providing the femurs.

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