Deep-Freezing Temperatures During Irradiation Preserves the Compressive Strength of Human Cortical Bone Allografts: A Cadaver Study

Abstract

Background: Gamma irradiation, which minimizes the risk of infectious disease transmission when human bone allograft is used, has been found to negatively affect its biomechanical properties. However, in those studies, the deep-freezing temperature during irradiation was not necessarily maintained during transportation and sterilization, which may have affected the findings. Prior reports have also suggested that controlled deep freezing may mitigate the detrimental effects of irradiation on the mechanical properties of bone allograft.

Question/purpose: Does a controlled deep-freezing temperature during irradiation help preserve the compressive mechanical properties of human femoral cortical bone allografts?

Methods: Cortical bone cube samples, each measuring 64 mm3, were cut from the mid-diaphyseal midshaft of five fresh-frozen cadaver femurs (four male donors, mean [range] age at procurement 42 years [42 to 43]) and were allocated via block randomization into one of three experimental groups (with equal numbers of samples from each donor allocated into each group). Each experimental group consisted of 20 bone cube samples. Samples irradiated in dry ice were subjected to irradiation doses ranging from 26.7 kGy to 27.1 kGy (mean 26.9 kGy) at a deep-freezing temperature below -40°C (the recommended long-term storage temperature for allografts). Samples irradiated in gel ice underwent irradiation doses ranging from 26.2 kGy and 26.4 kGy (mean 26.3 kGy) in a freezing temperature range between -40°C and 0°C. Acting as controls, samples in a third group were not subjected to gamma irradiation. The mechanical properties (0.2% offset yield stress, ultimate compression stress, toughness, and the Young modulus) of samples from each group were subsequently evaluated via axial compression loading to failure along the long axis of the bone. The investigators were blinded to sample group during compression testing.

Results: The mean ultimate compression stress (84 ± 27 MPa versus 119 ± 31 MPa, mean difference 35 [95% CI 9 to 60]; p = 0.005) and toughness (3622 ± 1720 kJ/m3 versus 5854 ± 2900 kJ/m3, mean difference 2232 [95% CI 70 to 4394]; p = 0.009) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than in those irradiated at deep-freezing temperatures (below -40°C). The mean 0.2% offset yield stress (73 ± 28 MPa versus 109 ± 38 MPa, mean difference 36 [95% CI 11 to 60]; p = 0.002) and ultimate compression stress (84 ± 27 MPa versus 128 ± 40 MPa, mean difference 44 [95% CI 17 to 69]; p < 0.001) of samples irradiated at a higher temperature range (-40°C to 0°C) were lower than the nonirradiated control group samples. The mean 0.2% offset yield stress (73 ± 28 MPa versus 101 ± 28 MPa, mean difference 28 [95% CI 3 to 52]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to those irradiated at deep-freezing temperature. The mean toughness (3622 ± 1720 kJ/m3 versus 6231 ± 3410 kJ/m3, mean difference 2609 [95% CI 447 to 4771]; p = 0.02; effect size = 1.0 [95% CI 0.8 to 1.2]) of samples irradiated at higher temperature range (-40°C to 0°C) were no different with the numbers available to the non-irradiated control group samples. The mean 0.2% offset yield stress, ultimate compression stress, and toughness of samples irradiated in deep-freezing temperatures (below -40°C) were not different with the numbers available to the non-irradiated control group samples. The Young modulus was not different with the numbers available among the three groups.

Conclusion: In this study, maintenance of a deep-freezing temperature below -40°C, using dry ice as a cooling agent, consistently mitigated the adverse effects of irradiation on the monotonic-compression mechanical properties of human cortical bone tissue. Preserving the mechanical properties of a cortical allograft, when irradiated in a deep-freezing temperature, may have resulted from attenuation of the deleterious, indirect effects of gamma radiation on its collagen architecture in a frozen state. Immobilization of water molecules in this state prevents radiolysis and the subsequent generation of free radicals. This hypothesis was supported by an apparent loss of the protective effect when a range of higher freezing temperatures was used during irradiation.

Clinical relevance: Deep-freezing temperatures below -40°C during gamma irradiation may be a promising approach to better retain the native mechanical properties of cortical bone allografts. A further study of the effect of deep-freezing during gamma radiation sterilization on sterility and other important biomechanical properties of cortical bone (such as, tensile strength, fracture toughness, and fatigue) is needed to confirm these findings.

Fig. 1

This schematic diagram shows the bone cutting process. The five femurs were systematically cut from segments into cortical rings, then into quarters, and finally into 64-mm³ cubes.

Fig. 2

A-B This figure shows timelines of the irradiation process for the (A) irradiated in dry ice and (B) irradiated in gel ice samples. The temperatures at key points are noted, with time (in hours) expressed as the cumulative duration starting after the packaging process at Hour 0. Periods of irradiation are highlighted for both treatment groups.

Fig. 3

Temperature pattern graphs are shown for (A) the irradiated in dry ice and (B) the irradiated in gel ice samples. All samples of each group were irradiated simultaneously. Periods of irradiation are highlighted for both groups, with Xs indicating the beginning and end of the respective periods. The internal temperature for the irradiated in dry ice samples was consistent throughout the process until unpackaging at 23 hours, with a mean temperature during irradiation of -78.4°C ± 0.2°C. The temperature in the irradiated in gel ice group, however, showed a steady but nonlinear increase from an initial temperature of -71.6°C to -1.1°C at 26 hours, with a mean temperature during irradiation of -13.4°C ± 7.8°C.

Fig. 4

A-C These photographs show the compression testing set-up. (A) In the mechanical testing process, compression platens were secured to the adjustable upper and lower grips of the Instron and (B) an allograft cortical bone cube was placed on the lower plate. (C) Axial force generated by the load cell compressed the specimen to failure.

Fig. 5

This graph shows the stress-strain curve of a sample from the control group. (A) The yield stress (x), which indicates the limit of a sample’s elastic behavior and the beginning of its plastic behavior, was determined using the 0.2% offset method [4, 32, 45, 46]. (B) Ultimate compressive stress (asterisk), defined as the maximum amount of compression stress a material can resist without fracturing, was taken as the largest stress of each stress-strain curve. (C) The Young modulus was derived by analyzing the gradient of the steepest slope of the linear portion of the stress-strain curve, where the stress was proportional to the strain; the nonlinear toe region was excluded from analysis. (D) The toughness of a material, defined as its ability to absorb the maximum energy before the point of fracture, was calculated as the area under the entire stress-strain curve, limited on the x-axis by the point of fracture; MPa = megapascal.

Fig. 6

These graphs show the stress-strain curves plotted using the combined mean of stress and strain values of every sample (taken at intervals of one second) of the (A) control, (B) irradiated in dry ice, and (C) irradiated in gel ice experimental groups. The mean yield stress (x), ultimate compressive stress (asterisk), the Young modulus, and toughness of each experimental groups are shown; MPa = megapascal; GPa = gigapascal; kJ/m^{3 =} kilojoule per cubic meter.