The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 86, Issue 12

Scientific Articles | December 01, 2004

The Effect of Gamma Radiation Sterilization on the Fatigue Crack Propagation Resistance of Human Cortical Bone

Erika J. Mitchell, MD¹; Allison M. Stawarz, MS²; Ramazan Kayacan, PHD³; Clare M. Rimnac, PHD²

¹ Department of Orthopaedics, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-7222. E-mail address for E.J. Mitchell: erikajas@aol.com
² Musculoskeletal Mechanics and Materials Laboratories, Department of Mechanical and Aerospace Engineering, Case Western Reserve University, 10900 Euclid Avenue, 620 Glennan Building, Cleveland, OH 44106
³ Department of Mechanical Engineering, Suleyman Demirel University, 32260 Isparta, Turkey

View Disclosures and Other Information

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from National Institutes of Health Grant AG17171, the Allen Scholarship Foundation, and a National Science Foundation Graduate Fellowship. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Investigation performed at the Department of Orthopaedics, University Hospitals of Cleveland, Cleveland, and the Musculoskeletal Mechanics and Materials Laboratories, Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, Ohio

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J Bone Joint Surg Am, 2004 Dec 01;86(12):2648-2657

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Abstract

Background: Clinical evidence has suggested that the rate of fracture in allografts sterilized with gamma radiation may be higher than that in controls. Gamma radiation sterilization has been shown to affect the post-yield properties of bone but not the elastic modulus. Since most allograft fractures occur with subcritical loads during activities of daily living, it may be that the fatigue properties of irradiated allografts are diminished. In this study, the fatigue crack propagation behavior of cortical bone sterilized with gamma radiation was compared with that of gender and age-matched controls. We hypothesized that gamma radiation significantly reduces the resistance of cortical bone to fatigue crack growth.

Methods: Specimens for fatigue crack propagation testing were machined from four pairs of fresh-frozen human femora obtained from four individuals (a younger male, younger female, older male, and older female donor). Half of the specimens were sterilized with 31.7 kGy of gamma radiation. The specimens were cyclically loaded to failure in a servohydraulic testing system, and crack growth was monitored. The cyclic stress intensity factor and the fatigue crack growth rate were calculated to examine the kinetics of fatigue crack growth. Following testing, the damage zone around the fracture plane was analyzed histologically.

Results: The morphology and kinetics of crack growth in irradiated specimens differed from the control data. Overall, the irradiated bone was significantly less resistant to fatigue crack growth than was control tissue (p < 0.05). There was less microdamage associated with fracture in the irradiated specimens than in the control specimens, with the exception of the bone from the older female donor.

Conclusions: Gamma radiation sterilization significantly reduces the fatigue crack propagation resistance of cortical bone. Irradiated specimens also demonstrate a smaller amount of microdamage along the fracture plane. These findings may be due to ultrastructural alterations in the collagen matrix caused by radiation.

Clinical Relevance: This study suggests that, despite having pre-yield mechanical properties that are similar to those of nonirradiated bone, gamma-radiation-sterilized allograft may be more predisposed to fracture even under the subcritical loads that occur during the activities of daily living.

Figures in this Article

Large cortical allografts have been used for many years in the reconstruction of large osseous defects secondary to trauma, tumor resection, or joint reconstruction. Patients treated because of trauma or tumors are often young, and the reconstruction of large osseous defects secondary to the reconstructions are generally in weight-bearing bones. The goal is to restore the patient to his or her prior functional status, and so the allograft must eventually be able to withstand the loads that occur during the activities of daily living of an active individual. Complete incorporation of allograft bone may take several years, during which time the patient has resumed weight-bearing activities¹. It is thus important that the graft material, in conjunction with an appropriate fixation device, be able to sustain substantial loads as it becomes incorporated into the host skeleton.

Allograft fractures often occur under normal loading conditions and have been found to occur in 12% to 20% of all large allografts^2-5. The addition of stress-risers in allograft bone by way of screw-holes, intraoperative scratches or cracks, and postoperative physiologic resorption may predispose the allograft to fracture. Fracture is one of the most frequent complications in the use of large cortical allografts in orthopaedic reconstructive surgery and carries substantial morbidity, often requiring additional surgery and in some cases even amputation^3-5.

Another risk associated with allograft implantation is infection from the cadaveric material. Concerns regarding allograft contamination with viruses such as the human immunodeficiency virus and hepatitis-C have prompted the use of gamma radiation sterilization. Radiation sterilization guarantees penetration throughout the allograft tissue and is relatively economical^6-8. However, in a clinical study, Lietman et al.³ found that, although grafts that were sterilized by gamma radiation showed a trend toward a lower infection rate than did nonirradiated grafts, they were significantly more likely to fracture (p = 0.03). In that study, 38% of the twenty-four grafting procedures performed with irradiated grafts were complicated by fracture, as opposed to 18% of the 282 nonirradiated grafts. The authors further showed that the prevalence of fracture was unrelated to the prevalence of nonunion. This result suggests that the increased prevalence of fracture in irradiated bone is not due to altered osteoinductive properties but rather may be due to changes in the mechanical properties of the graft material itself³.

Numerous studies have shown that the yield and post-yield mechanical properties of allograft bone are substantially altered by gamma radiation sterilization^3,9-12. Furthermore, the altered properties are dose-dependent^12,13. Mechanical tests have shown that gamma-irradiated cortical bone has decreased yield stress, ultimate strain, toughness, and work to fracture. However, the pre-yield properties, specifically the elastic modulus, were unchanged^9-12. Most allograft fractures occur under normal loading conditions where loads are within the pre-yield elastic range. Thus, other factors such as fatigue fracture resistance and the presence of defects or other stress-risers may be important fracture determinants.

Allograft fractures in both irradiated and nonirradiated bone tend to occur around stress-risers such as screw-holes or other defects created at the time of implantation^5,14,15. Fractures can occur even at loads within the pre-yield elastic range under these conditions because, in the vicinity of a defect, the stresses can exceed the yield stress. Akkus and Rimnac⁹ reported that the fracture toughness of human cortical bone sterilized with 27.5 kGy of gamma radiation was significantly reduced compared with nonirradiated bone (p < 0.05). They also observed that microdamage associated with the growing macrocrack was reduced by gamma radiation sterilization. This finding suggests that microdamage formation ahead of the crack tip is an important mechanism by which bone resists fracture. Microdamage at the crack tip may absorb energy that otherwise would be available to propagate the macrocrack^16,17.

Although the static fracture toughness of cortical bone is reduced by radiation sterilization, the effect of gamma radiation on crack propagation from a stress-riser under cyclic loading conditions is unknown. Most loading during activities of daily living is repetitive and in the pre-yield elastic range. There may be changes in the mechanical properties of allograft bone as it becomes vascularized and incorporated into host bone. However, it has been shown that allograft bone does not fully incorporate into the host, suggesting that the allograft tissue and adjunctive fixation need to withstand the stresses of daily activities¹. Thus, understanding the resistance of radiation-sterilized human cortical bone allograft to fatigue crack propagation is important.

We previously examined the fatigue crack propagation behavior of fresh-frozen cortical bone from younger (less than forty-year-old) and older (greater than sixty-year-old) male and female individuals¹⁸. In the present study, we hypothesized that gamma radiation sterilization significantly reduces the fatigue crack propagation resistance of human cortical bone. To test this hypothesis, we performed fatigue crack propagation tests on gamma-radiation-sterilized human cortical bone using the same cadaveric materials from the prior study to evaluate the effect of gamma radiation on the fatigue crack propagation resistance of human bone in the presence of a stress-riser.

Materials and Methods

Materials and Methods | Results | Discussion | References

Specimen Preparation

Four pairs of human femora with no known skeletal abnormality were obtained from the Musculoskeletal Transplant Foundation (Edison, New Jersey) and the Anatomical Donations Program at the University of Michigan Medical School (Ann Arbor, Michigan). Femora were obtained from one younger (fifteen-year-old) female, one younger (eighteenyear-old) male, one older (sixty-one-year-old) male, and one older (seventy-five-year-old) female donor (Table I). Each femur was stripped of soft tissue, and the mid-portion of the diaphysis was sectioned into rings approximately 30 mm in length with use of an autopsy saw (Stryker, Rutherford, New Jersey). Each ring was then wet-machined with use of a tabletop micromill (Micromill 2000; Denford, Medina, Ohio) that was numerically controlled by a computer to produce notched compact tension specimens that were 17.5 mm wide, 16.8 mm high, and 3.0 mm thick from the medial and lateral cortices (Figs. 1-A and 1-B)¹⁹. The specimens were machined such that crack growth would occur parallel to the long axis of the femur. Although fatigue fractures most commonly occur in the transverse plane, across osteons, rather than along them, longitudinal fractures have been reported²⁰. The notch was cut into each specimen with a low-speed diamond saw and was razor-sharpened with a microtome 250 µm beyond the machined notch to serve as the initiating stress-riser for fatigue crack propagation. Four to nine compact tension specimens for fatigue crack propagation testing were machined from each femur (twenty-eight specimens were used as controls and twenty-two specimens were irradiated). The number of specimens from each femur was limited by the geometry and diameter of each bone such that fewer specimens were obtainable from the smaller, older female donor, for instance, than from the larger, younger male donor. Specimens were kept wet frozen until testing.

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TABLE I Fatigue Crack Propagation Resistance of the Test Groups Assessed with the Pooled Regression*

	Slope (m)					Constant (C)
Test Group	Control Specimens	Irradiated Specimens	P Value†	Control Specimens	Irradiated Specimens	P Value†
Bone from younger (18-yr-old) male donor	7.21 (0.34)	5.99 (0.28)	0.006‡	-8.42 (0.08)	-6.67 (0.04)	0.000‡
Bone from younger (15-yr-old) female donor	5.90 (0.38)	4.73 (0.32)	0.019‡	-7.82 (0.10)	-6.35 (0.05)	0.000‡
Bone from older (61-yr-old) male donor	5.89 (0.47)	4.94 (0.28)	0.066	-7.24 (0.10)	-6.21 (0.04)	0.000‡
Bone from older (75-yr-old) female donor	5.60 (0.73)	3.74 (1.27)	0.171	-6.61 (0.12)	-6.20 (0.17)	0.042‡

The values are given as the mean (and standard error) for the slope (m) and for the constant (C) for the pooled regression for each of the test groups, with use of the relationship: log(da/dN) = log(C) + mlog(?K), where da/dN is the rate of fatigue crack propagation and ?K is the cyclic stress intensity factor. †P values refer to a comparison of the control versus the irradiated condition for each age-gender test group.Significance taken at p < 0.05.

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TABLE I Fatigue Crack Propagation Resistance of the Test Groups Assessed with the Pooled Regression*

	Slope (m)					Constant (C)
Test Group	Control Specimens	Irradiated Specimens	P Value†	Control Specimens	Irradiated Specimens	P Value†
Bone from younger (18-yr-old) male donor	7.21 (0.34)	5.99 (0.28)	0.006‡	-8.42 (0.08)	-6.67 (0.04)	0.000‡
Bone from younger (15-yr-old) female donor	5.90 (0.38)	4.73 (0.32)	0.019‡	-7.82 (0.10)	-6.35 (0.05)	0.000‡
Bone from older (61-yr-old) male donor	5.89 (0.47)	4.94 (0.28)	0.066	-7.24 (0.10)	-6.21 (0.04)	0.000‡
Bone from older (75-yr-old) female donor	5.60 (0.73)	3.74 (1.27)	0.171	-6.61 (0.12)	-6.20 (0.17)	0.042‡

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Fig. 1-A: Schema of a compact tension specimen as machined from the femoral diaphyseal cortex.

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Fig. 1-B: Schema of a compact tension specimen with dimensions.

For each age-gender pair of femora, there were two groups: control specimens (fresh-frozen only) and gammaradiation-sterilized specimens. Specimens were assigned to the radiation test group or to the control test group from either the right or the left femur of each pair of femora. In addition, the assignments were made such that specimens from the same diaphyseal location of left and right femoral pairs served as matched pairs. The radiation test group specimens were kept wet frozen, were packed in polyethylene bags, and were surrounded with dry ice for shipment for gamma radiation sterilization by a commercial sterilizing vendor (STERIS Isomedix, Groveport, Ohio). The specimens were kept frozen on dry ice throughout the process to minimize the formation of free radicals in the collagen matrix²¹. Specimens were irradiated in an oxygen environment in two batches and received an average dose of 31.7 kGy (range, 29.6 to 33.5 kGy). The recommended dose for allograft sterilization is 25 to 35 kGy^6-8.

Fatigue Crack Propagation

Fatigue crack propagation tests were conducted with use of the American Society for Testing and Materials standard E 647-95a, "Standard Test Method for Measurement of Fatigue Crack Growth Rates," as a guide¹⁹. All tests were conducted on a closed-loop, servohydraulic testing machine (Instron, Canton, Massachusetts). A sinusoidal waveform with a cyclic frequency of 2 Hz and an R-ratio (ratio of minimum load to maximum load) of 0.1 was used. A continuous, heated water drip that was monitored with a thermocouple kept the specimens moist and at 37°C throughout testing. To improve visualization of the crack tip, the specimen surfaces were hand polished with polishing compounds that had a progressively finer grit, finishing with a grit size of 0.05 µm. Crack length was monitored and was measured periodically with use of a traveling microscope (Gaertner Scientific, Skokie, Illinois) on an x-y stage. The resolution of the x-y stage micrometer was 10 µm. In addition, during testing, the surface was darkened with pencil graphite to enhance visualization of the crack tip. Stable fatigue crack growth was monitored on each specimen until catastrophic failure occurred. If there was crack branching or if there were two or three cracks ahead of the notch, the crack length was measured as the farthest crack tip from the load-line.

Fatigue crack growth behavior was also observed during testing. That is, features such as crack bifurcation, growth into pores or vascular channels, deformation ahead of the crack tip, and bridging of fracture surfaces behind the crack tip by lamellar fibrils were noted¹⁸.

For each specimen, the rate of fatigue crack propagation, da/dN (mm/cycle), and the cyclic stress intensity factor, ?K (Mpa[m]^1/2) were determined according to established methods¹⁹. The cyclic stress intensity factor is a measure of the amplification of stress at the crack tip and is a function of specimen geometry, crack length, and cyclic load²². For a constant cyclic stress, the cyclic stress intensity factor and the rate of fatigue crack propagation increase as the crack length increases. It has been shown that the resistance of a material to fatigue crack propagation can be evaluated by examining the log(da/dN) versus log(?K) relationship²². In a comparison of two treatment groups or conditions at the same cyclic stress intensity factor (?K), the treatment group with the higher rate of fatigue crack propagation (da/dN) is considered to have a lower resistance to fatigue crack propagation because the crack is growing faster.

To compare fatigue crack propagation resistance between treatment groups, the data from all specimens for a given age-gender group in both the control and the irradiated condition were pooled and linear regression analysis of log(da/dN) versus log(?K) was determined with the formula: log(da/dN) = log(C) + mlog(?K).

The constant (C) and the slope (m) are a function of material properties and test conditions²². Note that the constant is the fatigue crack growth rate at ?K = 1.0 MPa(m)^1/2. A significant difference in the constants between two groups is indicative of a change in fatigue crack propagation resistance. The group with the higher constant has lower resistance to fatigue crack propagation. A significant difference in the slope between two groups is indicative of a change in fatigue crack growth mechanisms at the crack tip. An increase in the slope indicates that a material is more sensitive to changes in the cyclic stress intensity factor (?K).

The relative fatigue crack propagation resistance between groups was considered with respect to differences in the constant (C) and the slope (m). Comparisons of the slope and the constant of the regression lines were made, with use of the linear test method²³, between the control and irradiated conditions in each age-gender group: control versus irradiated specimens from the younger male group, control versus irradiated specimens from the younger female group, control versus irradiated specimens from the older male group, and control versus irradiated specimens from the older female group. A p value of <0.05 was considered significant. In addition, the percentage change in the mean constant between the irradiated and control groups was examined to consider the relative magnitude of change in the fatigue crack propagation resistance between the control and irradiated conditions for each age-gender group.

Wet Density and Ash Weight Analysis

Tissue characterization of wet density and ash weight was performed following established methods to determine whether there were changes to the irradiated bone tissue that may have affected its fatigue crack propagation resistance²⁴. A piece of bone that was approximately 17.5 mm wide, 3.5 mm high, and 3 mm thick was cut off from the superior and inferior portion of each specimen for ash weight and density analysis. Twotailed t tests were performed to determine differences between control and irradiated tissue as well as differences between age-gender groups. A p value of <0.05 was considered significant.

Histological Analysis of Damage Along the Crack Front

Except for the portion used for tissue characterization, each tested specimen was bulk-stained in basic fuchsin and embedded in polymethylmethacrylate for sectioning in preparation for histological analysis following established methods²⁵. After staining, the embedded specimens were cut into 250-µm sections perpendicular to the plane of crack growth and parallel to the direction of crack growth (Fig. 2) with use of a low-speed diamond saw (Buehler, Lake Bluff, Illinois). The sections were mounted onto acrylic slides and were ground and polished to a 100-µm thickness. A section of each fracture specimen was analyzed for microdamage near the plane of fracture with use of a Bioquant Advanced Image Analysis system (2000 R and M Biometrics, Nashville, Tennessee) and epifluorescent microscopy with a Texas red filter. Under these lighting conditions, the zone of microdamage above and below the fracture plane appeared bright compared with the undamaged bone. This methodology has been used to identify microdamage in the vicinity of a fracture plane in bone tissue under static loading conditions²⁶. For each specimen, beginning at the notch, measurements of microdamage thickness, in micrometers, were made in 0.1-mm increments up to failure (Fig. 2). There were approximately forty measurements per specimen. Microdamage thickness versus cyclic stress intensity factor (?K) was pooled for each group of specimens, and comparisons were made between the control and irradiated groups. Differences in the slope and the constant between groups were examined with use of the linear test method (p < 0.05).

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Fig. 2: Schema of one-half of a compact tension specimen after testing, demonstrating a region of microdamage below the fracture plane. The thickness of the microdamage was determined at selected crack lengths.

Results

Materials and Methods | Results | Discussion | References

Fatigue Crack Propagation Resistance

Seven specimens were lost during testing because of machine problems. For the forty-three other specimens, stable fatigue crack propagation was achieved for all age-gender groups in both the control and the radiation-sterilized condition. The fatigue crack propagation resistance of human cortical bone was significantly reduced by gamma radiation sterilization in all four test groups (p < 0.05; Figs. 3-A through 3-D) as determined by a significant increase in the constant (C) (Table I). The percentage difference of the means of the constant between different treatment groups demonstrated that the loss in fatigue crack propagation resistance between control and irradiated specimens was greatest in the younger male group, followed by the younger female group, then the older male group (Table II). However, despite having the largest loss in fatigue crack propagation resistance, the younger male group had an end result after radiation that was similar to the fatigue resistance of the other groups after radiation (Table I). The older female specimens had the worst fatigue crack propagation resistance of the four control age-gender groups and had the smallest loss in fatigue crack propagation resistance after radiation (Table II). The slope (m) of each regression was significantly decreased by irradiation in the younger male and younger female groups and was not significantly altered in the older male and older female groups (Table I). However, the trend for all groups was toward a lower slope after irradiation.

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Figs. 3-A through 3-D: Comparison of the fatigue crack growth rate (da/dN) versus the cyclic stress intensity factor (?K) in the control and gamma-radiation-sterilized conditions. Note that regression lines have been extended beyond the data points for clarity. Fig. 3-A Bone from the younger male donor. Fig. 3-B Bone from the younger female donor.

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Fig. 3-C: Bone from the older male donor. Fig. 3-D Bone from the older female donor.

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TABLE II Percentage Difference of the Means of the Constants for the Irradiated Specimens Compared with the Control Specimens for Each Age-Gender Group*

Test Group	Percentage Difference in Constants
Bone from younger male donor	20.78
Bone from younger female donor	18.80
Bone from older male donor	14.23
Bone from older female donor	6.20

The greatest change in fatigue crack propagation resistance occurred in the younger male group, and the least change occurred in the older female group.

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TABLE II Percentage Difference of the Means of the Constants for the Irradiated Specimens Compared with the Control Specimens for Each Age-Gender Group*

Test Group	Percentage Difference in Constants
Bone from younger male donor	20.78
Bone from younger female donor	18.80
Bone from older male donor	14.23
Bone from older female donor	6.20

The greatest change in fatigue crack propagation resistance occurred in the younger male group, and the least change occurred in the older female group.

Crack Propagation Observations

For both the control and the irradiated age-gender specimen groups, fatigue crack growth proceeded such that there were periods of stable crack growth followed by periods of crack arrest. This resulted in a discontinuous, acceleration-deceleration pattern of crack growth¹⁸. During cycling, a region of deformation was observed to develop ahead of the crack tip in both the control and the irradiated specimens, during which time the crack tip would not advance. The region would become increasingly prominent with cycling until there was an increment of crack tip advance through the region and the process would begin again. In control specimens, the crack often bifurcated or diverted, which was, in some cases, observed around microstructural barriers such as osteons or along cement lines. When this occurred, crack growth was also seen to decelerate. One of the bifurcated cracks would eventually become the dominant fatigue crack leading to failure. Crack bifurcation was never observed in the irradiated specimens. Crack growth was also observed to decelerate when the crack encountered a pore or vascular channel in specimens from younger bone in both the control and irradiated conditions. In comparison, crack growth into a pore or vascular channel in specimens from older bone, in both the control and the irradiated condition, often led to catastrophic failure. In all groups, the end of stable fatigue crack growth was marked by the development of multiple cracks rapidly growing across the specimen, leading to catastrophic failure.

In video analysis of behavior at the crack tip, lamellar fibrils were sometimes observed in the control specimens behind the crack tip, bridging the two fracture surfaces, as has been reported¹⁸. These lamellar fibril bridges eventually fractured as the crack continued to propagate. Lamellar fibril bridges were not observed in the irradiated specimens.

Wet Density and Ash Weight Analysis

No significant difference in the wet density or ash weight was detected between control and irradiated material for any age-gender group (Table III). In a comparison of age-gender groups, specimens from the older female group had a significantly lower wet density and ash weight than did specimens from the younger female group (p < 0.05).

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TABLE III Comparison of the Wet Densities and Ash Contents for Each Treatment Group*

	Control Specimens	Irradiated Specimens	P Value
Wet density (g/mL)
Younger male group	1.990 (0.0145)	1.989 (0.0120)	0.929
Younger female group	1.993 (0.0111)	1.982 (0.0144)	0.211
Older male group	2.014 (0.0070)	2.014 (0.0089)	0.976
Older female group	1.978 (0.0064)	1.945 (0.0659)	0.381
Ash content (% dry wt)
Younger male group	0.5895 (0.0407)	0.6112 (0.0090)	0.219
Younger female group	0.6147 (0.0121)	0.6081 (0.0203)	0.526
Older male group	0.6099 (0.0508)	0.5887 (0.0494)	0.505
Older female group	0.5944 (0.0019)	0.6065 (0.0096)	0.089

The values are given as the mean (and the standard deviation). No significant differences were detected between any of the irradiated and control specimen groups.

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TABLE III Comparison of the Wet Densities and Ash Contents for Each Treatment Group*

	Control Specimens	Irradiated Specimens	P Value
Wet density (g/mL)
Younger male group	1.990 (0.0145)	1.989 (0.0120)	0.929
Younger female group	1.993 (0.0111)	1.982 (0.0144)	0.211
Older male group	2.014 (0.0070)	2.014 (0.0089)	0.976
Older female group	1.978 (0.0064)	1.945 (0.0659)	0.381
Ash content (% dry wt)
Younger male group	0.5895 (0.0407)	0.6112 (0.0090)	0.219
Younger female group	0.6147 (0.0121)	0.6081 (0.0203)	0.526
Older male group	0.6099 (0.0508)	0.5887 (0.0494)	0.505
Older female group	0.5944 (0.0019)	0.6065 (0.0096)	0.089

The values are given as the mean (and the standard deviation). No significant differences were detected between any of the irradiated and control specimen groups.

Microdamage Analysis

In all but the older female group, microdamage thickness increased with increasing cyclic stress intensity factor (?K) (Figs. 4-A through 4-D). For the younger male, younger female, and older male groups, a significant difference in microdamage thickness versus the cyclic stress intensity factor was detected between the control and irradiated conditions, as demonstrated by a significantly higher slope (Table IV). Interestingly, the slopes for microdamage thickness versus the cyclic stress intensity factor for both the control and irradiated older female groups were close to 0 and were not significantly different. Overall, for all but the older female group, the gamma-irradiated bone groups demonstrated less microdamage at a given cyclic stress intensity factor than did the control groups. Also, the range of microdamage thickness was smaller for the irradiated bone groups (2 to 123 m) than for the nonirradiated bone groups (4 to 352 m).

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Figs. 4-A through 4-D: Comparison of microdamage thickness versus the cyclic stress intensity factor (?K) in the control and gamma-radiation-sterilized conditions. Fig. 4-A Bone from the younger male donor. Fig. 4-B Bone from the younger female donor.

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Fig. 4-C: Bone from the older male donor. Fig. 4-D Bone from the older female donor.

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TABLE IV Microdamage Thickness Versus Cyclic Stress Intensity Factor (?K) for the Pooled Regression for Each Test Group*

	Slope (m)					Constant (C)
Test Group	Control Specimens	Irradiated Specimens	P Value†	Control Specimens	Irradiated Specimens	P Value†
Bone from younger male donor	1.32 (0.13)	0.55 (0.10)	0.000‡	1.44 (0.04)	1.56 (0.02)	0.047‡
Bone from younger female donor	1.36 (0.14)	0.48 (0.10)	0.000‡	1.50 (0.04)	1.55 (0.02)	0.196
Bone from older male donor	1.12 (0.15)	0.59 (0.12)	0.005‡	1.47 (0.04)	1.52 (0.02)	0.208
Bone from older female donor	-0.20 (0.17)	0.19 (0.14)	0.133	1.63 (0.04)	1.44 (0.02)	0.000‡

The values are given as the mean (and the standard error). †P values refer to a comparison of the control versus irradiated conditions for each age-gender test group. ‡Denotes significance at p < 0.05.

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TABLE IV Microdamage Thickness Versus Cyclic Stress Intensity Factor (?K) for the Pooled Regression for Each Test Group*

	Slope (m)					Constant (C)
Test Group	Control Specimens	Irradiated Specimens	P Value†	Control Specimens	Irradiated Specimens	P Value†
Bone from younger male donor	1.32 (0.13)	0.55 (0.10)	0.000‡	1.44 (0.04)	1.56 (0.02)	0.047‡
Bone from younger female donor	1.36 (0.14)	0.48 (0.10)	0.000‡	1.50 (0.04)	1.55 (0.02)	0.196
Bone from older male donor	1.12 (0.15)	0.59 (0.12)	0.005‡	1.47 (0.04)	1.52 (0.02)	0.208
Bone from older female donor	-0.20 (0.17)	0.19 (0.14)	0.133	1.63 (0.04)	1.44 (0.02)	0.000‡

Discussion

Materials and Methods | Results | Discussion | References

This investigation is one of the first studies to examine how human cortical bone tissue resists fatigue crack growth. In addition, this study examined the relative fatigue crack growth behavior of macrocracks rather than of microcracks. The growth of macrocracks is clinically relevant in terms of the development of stress fractures. This study demonstrates that cracks can be grown experimentally in human cortical bone in a stable manner under cyclic loading conditions. It was found that gamma radiation sterilization led to a significant decrease in fatigue crack propagation resistance. In addition, it was observed that there was less crack branching and less fibril bridging behind the crack tip in the gamma-irradiated bone groups compared with the nonirradiated bone groups. The compositional tissue characteristics of wet density and ash weight were unchanged following irradiation. It is thus postulated that the differences in fatigue crack propagation resistance between nonirradiated and irradiated bone tissue in this study were almost certainly due to structural changes in the bone tissue caused by gamma radiation rather than to alterations in mineral or organic content.

Interestingly, the effect of radiation on fatigue crack propagation resistance appeared to be influenced by the age and gender of the donor. The greatest loss of fatigue crack propagation resistance caused by radiation was found in bone from the younger male donor, whereas the smallest loss in fatigue crack propagation resistance was found in bone from the older female donor (Table II), but the irradiated fatigue crack propagation resistance was similar for both groups. These results may be due to the fact that both radiation sterilization and aging are known to affect the ultrastructure of the bone collagen matrix. Gamma radiation in doses as low as 10 kGy is known to cause cross-linking and scission in the collagen matrix^3,13. Cheung et al.¹³ found that, with gamma radiation, alpha chains in the collagen helix are cleaved in a dose-dependent manner. In collagen specimens treated with 10 kGy of gamma radiation, approximately 5% of the alpha chains were degraded. At 75 kGy, up to 40% of the chains were smaller than their intact counterparts¹³. These ultrastructural changes have been correlated with decreased fracture strength as determined by three-point bending tests^11,21. Alterations in collagen cross-linking are also known to occur with aging^10,13,21,27. There is a higher ratio of stable cross-links to reducible cross-links in older bone compared with younger bone^27,28. Given this, it is possible that gamma radiation may have less of an effect on the collagen structure of older bone because there are fewer reducible cross-links in older bone compared with younger bone.

The formation of microdamage at a crack tip in bone tissue results in energy dissipation and has been shown to be an important mechanism by which bone tissue inhibits crack growth under static loading conditions⁹. The acceleratingdecelerating behavior of crack growth that was noted in this study may also relate to the formation of microdamage. Vashishth et al.¹⁷ developed a model of microdamage and crack growth from cyclically loaded specimens of cortical bone. In that model, a microcrack zone forms around the crack tip. With continued loading, the crack grows through this microdamage zone, accelerating into undamaged bone. The rate of crack propagation then decreases until another microdamage zone is formed at the new crack tip¹⁷. Our data showed that less microdamage is formed during crack growth in irradiated specimens than in controls. Furthermore, the least damage occurred in the control and irradiated specimens from the older female group, which had the least resistance to fatigue crack propagation.

It is hypothesized that the changes in the collagen ultrastructure due to gamma radiation and aging inhibit the formation of microdamage at the crack tip, which, in turn, facilitates propagation of a fatigue crack. In support of this hypothesis, our results showed that there was less microdamage around a crack in irradiated specimens than in control specimens (with the exception of the older female specimens) and less microdamage in older bone than in younger bone. In this study, the smallest loss of fatigue crack propagation resistance with gamma radiation sterilization was seen in the older female group. These findings suggest that ultrastructural changes to the collagen caused by aging or by gamma radiation sterilization create comparable losses in the resistance to fatigue crack propagation.

We speculate that crack branching and the formation of lamellar fibrils bridging the fracture surfaces behind the crack tip also are mechanisms that contribute to inhibiting fatigue crack propagation in bone tissue¹⁸. A similar fracture resistance mechanism has been observed in dentine²⁹. The lack of crack branching and lamellar bridging in irradiated bone suggests that these mechanisms of inhibiting crack growth are altered by gamma radiation, contributing to the decrease in fatigue crack propagation resistance.

A limitation of this study is that only one pair of femora was studied for each age-gender group; thus, the results regarding age and gender differences on fatigue crack propagation resistance can only be considered as suggestive at this time. However, in our comparison of irradiated and nonirradiated bone, each irradiated specimen had a control matched for diaphyseal location from the contralateral femur. Furthermore, both groups included specimens from both the left and the right femur in each pair. Therefore, within each femoral pair, multiple specimens were tested and controlled for location, age, gender, and loading history. This allowed us to examine specifically the effects of irradiation on fatigue crack propagation resistance in comparison with a similar nonirradiated specimen of human bone. Another possible limitation of the present study is that it was an in vitro, not an in vivo, study; the fatigue crack propagation behavior of bone tissue in vivo may be different from that which occurs in vitro. These in vitro results also represent, at best, "time-zero" conditions, and it is possible that there would be some change in fatigue crack propagation resistance with graft incorporation. However, it has been shown that allograft bone does not fully incorporate into the host bone¹, thus, comparison of in vitro fatigue crack growth resistance is still likely to be clinically relevant for allograft bone.

It is difficult to ascertain the threshold at which the difference in fatigue crack propagation resistance between irradiated and nonirradiated allograft bone is clinically important. Bone from elderly donors is already often excluded from tissue banking because of presumed fragility. Although our sample size was inadequate for a thorough examination of fatigue crack propagation resistance in different age-groups, our data demonstrated similar crack growth rates in bone that had been irradiated and in bone specimens from older donors.

In the present study, fatigue crack propagation resistance was evaluated along the longitudinal axis of bone. Clinically, fatigue fractures most commonly occur in the transverse plane, across osteons, rather than along them, although longitudinal fractures have been reported²⁰. It would be of interest to examine the fatigue crack propagation resistance of bone transverse to the osteons. Initial efforts to study fatigue crack growth transversely across osteons have been met with limited success to date, since there is a tendency for the crack to deviate out of plane, as has been observed under static loading conditions⁹. However, efforts are underway to use grooved specimens to sustain fatigue crack growth in the transverse plane.

We have shown in this study that the fatigue crack propagation resistance of cortical bone sterilized with gamma radiation is significantly decreased compared with age-matched controls. This may explain the high prevalence of fracture in large osseous allografts that have been irradiated³ despite reports that the pre-yield elastic properties are unchanged^11,12. Clinically, the results of this study suggest that intraoperative precautions should be taken to reduce the number of stress-risers in gamma-radiation-sterilized allograft tissue to inhibit the formation of fatigue cracks. This includes avoiding scratches in the allograft bone or choosing intramedullary fixation where possible to avoid the introduction of multiple screw-holes. Alternatively, the surgeon may opt for allograft bone sterilized by other means. ?

References

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Topics

fatigue ; fracture ; crack cocaine ; gamma rays ; tooth fractures ; sterilization for infection control ; compact bone

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Erika J. Mitchell, Allison M. Stawarz, Ramazan Kayacan, Clare M. Rimnac; The Effect of Gamma Radiation Sterilization on the Fatigue Crack Propagation Resistance of Human Cortical Bone. The Journal of Bone & Joint Surgery. 2004 Dec;86(12):2648-2657.

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