Time-dependent changes in dynamic mechanical properties of irradiated bone

Abstract

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. The currently available literature contains few examples of studies on long-lasting strains of bones but no example for irradiated bones. In this study the bovine femurs from a 2-year-old animal were machine cut and irradiated with the doses of 10, 15, 25, 35, 45 and 50 kGy. The dynamic mechanical analysis was performed at 1 Hz at the room temperature in a 3-point bending configuration for 2880 minutes. The final values of $E^{'}$ and $E^{″}$ were dose independent but they were reached at different periods. For this reason, so called “critical point” was introduced for the further analysis. All the examined sample groups were characterized by statistically significant lower values of the critical point in comparison with the control samples ( $p < 0.05$ ) but the biggest differences were observed between the control samples and the samples irradiated with the doses of 10, 15 and 25 kGy. Current results and literature review suggest that the dose of 35 kGy is the optimal dose for ionizing radiation sterilization.

Keywords

Bone radiation sterilization DMA (Dynamic Mechanical Analysis)

1. Background

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques [1]. Ionizing radiation (β or γ) is one of the most widespread methods of bone sterilization due to very good penetration and high efficacy in microorganisms inactivation [2,3]. So far no international consensus concerning the optimal radiation dose has been reached, therefore the recommended dose in different countries vary between 15–35 kGy [4]. In Poland the dose of 35 kGy is normally used in the National Center for Tissue and Cell Banking in Warsaw [5]. In comparison, the dose of 25 kGy is the gold standard in the US [6]. A new bone is formed based on the transplanted bone tissue and this process is related with increased activity of osteoclasts, which initiate the process of bone trabeculae resorption. The remodeling of a transplanted bone lasts from a few months to a few years, depending on the size of the transplant, its physical and biological properties and the general condition of a patient [7–10]. Therefore it is so important that the bone preserve its mechanical properties.

The influence of ionizing radiation on mechanical properties of the bones was raised in a number of research [11–15], mainly with the use of static testing. Research showed that radiation sterilization, even with high doses, do not affect the mechanical properties of the bones measured with Young’s modulus, however it significantly deteriorates other parameters such as: work to fracture, bending strength or compressive strength [11–15].

So far the influence of ionizing radiation on the dynamic properties of bones has only been discussed in the last work published by the authors [16]. This research indicates that the samples irradiated with the dose of 25 and 35 kGy are the closest to the control samples, what suggests the doses applied so far in sterilization both in Poland and the United States are fully justified [16]. Unfortunately there were no answer witch dose, 25 or 35 kGy is superior one. Therefore, further research was initiated to answer to the phenomenon described above. Particularly due to the fact, that currently available literature contains few examples of studies on long-lasting strains of biological materials, including the dynamic mechanical analysis (DMA) applied for bones. In the work by Yamashita et al. [17] isothermal tests were carried out at 37°C for only 150 minutes. A steep increase of $E^{'}$ values detected in the first 20 min was followed by a slower rise, and moreover, the experiment involved a non-radiated bone. Nevertheless, there is no available literature concerning long-lasting strains of a bone exposed to ionizing radiation, what is significant information due to the time of bone remodeling.

2. Materials and methods

2.1. Samples preparation

Bovine femurs from a 2-year-old animal have been used for this research. To minimize the influence of biological variability on the analyzed material, bones from one animal have been used. After mechanical cleaning, the middle part of the corpus femur made of compact bone has been chosen for further treatment. Cuboidal samples with the parameters 30 × 5 × 2 mm have been extracted from the bone shaft with a diamond saw. The longest parameter is parallel to long bone axis. The samples have been ground to obtain smooth, flat parallel surface. During the cutting and grinding processes, the bone was being cooled with water to avoid temperature increase and thermal denaturation of collagen. After drying at room temperature each sample was packed separately in a disposable container made from PET foil. Research protocol was approved by the Department of Biophysics Advisory Board, consent of Bioethical Committee was not needed because material used in this study was designed for comestible purposes.

2.2. Irradiation

The samples were irradiated with an electron beam of the energy 10 MeV, with a given current of 550 mA in the Centre for Radiation Research and Technology, Institute of Nuclear Chemistry and Technology in Warsaw, Poland. The samples were divided into 7 groups depending of the radiation dose: 0 (control), 10, 15, 25, 35, 45 and 50 kGy (2 samples in every group).

2.3. DMA testing

DMA examination was carried out by means of the DMA 242 analyzer made by Netzsch, at the frequency of 1 Hz, applying the 3-point bending configuration, establishing the complex modulus with the following equation: $E^{*} = \frac{l^{3}}{4 b h^{3}} \cdot \frac{F}{a^{*}},$ (1) where: $E^{*}$ – complex modulus [Pa], l – sample length [mm], b – wideness [mm], h – sample height [mm], F – strength [Pa], $a^{*}$ – total dynamic displacement [mm].

From the complex modulus ( $E^{*}$ ), storage modulus ( $E^{'}$ ) and loss modulus ( $E^{″}$ ) were assessed $E^{*} = E^{'} + i E^{″} .$ (2)

The pressure strength equals 1 N. Experiment time was assumed for 2880 minutes in controlled room temperature (25°C).

2.4. Statistical analysis

The obtained results of the mechanical examination have been processed in a digital form, illustrated and analyzed with the software Origin Pro 7.0. The statistical analysis was performed with Statistica 10 package. Friedman tests was applied for the comparisons between the groups, assuming the confidence level of $α = 0.05$ .

Fig. 1.

Time dependent changes in $E^{'}$ and $E^{″}$ : (a) control samples; (b) samples irradiated with the dose of 10 kGy; (c) samples irradiated with the dose of 15 kGy; (d) samples irradiated with the dose of 25 kGy; (e) samples irradiated with the dose of 35 kGy; (f) samples irradiated with the dose of 45 kGy; (g) samples irradiated with the dose of 50 kGy.

Fig. 1.

(Continued.)

3. Results and discussion

In the experiment presented herein, the samples were exposed to long-lasting (48 hours) force of the value of 1 N, operating with the frequency of 1 Hz in room temperature. The frequency of 1 Hz is most frequently used for this type of DMA research, and according to Mano et al. [18] it corresponds best with the physiological conditions. The obtained results are presented in Fig. 1. In all cases, a rapid increase of $E^{'}$ and $E^{″}$ values was observed within the first 100–150 minutes of the research. Following that, $E^{'}$ and $E^{″}$ slowly increased, reaching a type of plateau. A relative stabilization of $E^{'}$ and $E^{″}$ values, depending on the dose, was observed after approximately 300–800 minutes. No data are available on long-lasting duration of the experiment in papers on dynamic mechanical properties of bone, excluding Yamashita et al. [17]. Even though, in the above mentioned experiment the duration of the test was limited to 150 minutes and the highest increase of $E^{'}$ value was noticed in the first 20 minutes.

Fig. 2.

Method using retardation time to indicate the “critical point”.

Fig. 3.

Influence of irradiation on dynamic mechanical properties of bone according to “critical point”.

In the experiment presented herein, the final values of $E^{'}$ and $E^{″}$ were dose independent and the plateau was reached at different periods. For this reason, so called “critical point” was introduced for the further analysis. From the typical curve $E^{'} = f (t)$ “critical point” was determined in a similar way as the retardation time, when the $E^{'}$ values reached the 67% of the plateau (Fig. 2). Following that, the values for particular doses were presented in Fig. 3. It was stated, that all the examined sample groups are characterized by statistically important lower values of the critical point in comparison with the control samples ( $p < 0.05$ ). The biggest differences were observed between the control samples and the samples irradiated with the doses of 10, 15 and 25 kGy. The smallest differences were observed between the control samples and the sample groups irradiated with following doses: 35, 45 and 50 kGy. These results suggest that influence of ionizing radiation on bone mechanical properties is not a simple linear and are contrary to research, which use the static tests, where the mechanical properties are deteriorating with the increase of the dose [1,12,13,15,19]. In the work by Mardas et al. [16], describing the influence of ionizing radiation on dynamic properties of the bone, it was shown that within the dose 10–50 kGy, the most similar mechanical properties to control samples were those irradiated with 25 and 35 kGy. The analysis of the long-lasting DMA supports the doses of 35, 45 and 50 kGy. Only one dose in current experiment and other with DMA testing [16] in common – the dose of 35 kGy witch seems to be the optimal one and should be the suggested dose of ionizing radiation applied in bone sterilization.

The clinical research also confirms the effectiveness of the bones transplanted which were irradiated with the doses of 25 and 35 kGy. The study carried out by Marczyński et al. [20] on over 1100 patients receiving transplants of a frozen bone irradiated with the dose of 35 kGy demonstrated a very good treatment results in 83% of cases, good results in 10%, satisfactory results in 6% and unsatisfactory results in only 1% of cases. During a 3-year observation of 127 patients after massive bone transplants Hernigou et al. [21] showed that using the dose of 25 kGy for sterilization brings complications in the form of fractures at the level of 6%, or non-union bone at the level of only 5.5%. This result did not vary much from the research of a non-radiated bone. Also in the research of Loty et al. [3], where clinical analysis of 150 massive transplants were carried out, it was showed that there were no significant differences between the bones radiated with the dose of 25 kGy, and non-radiated bones.

4. Conclusions

Based on the obtained results, it was stated that the doses of 35, 45 and 50 kGy in the long-lasting DMA analysis are more similar to the control samples than the doses of 10, 15 and 25 kGy. Current results and literature review suggest that the dose of 35 kGy is the optimal dose for ionizing radiation sterilization. The application of an additional parameter, the critical point, based on the calculated retardation time may be useful during the strength tests of biological materials.

Footnotes

Acknowledgement

Thanks to Arek Piechocki for language corrections of this manuscript.

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