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Radiation sterilized bone response to dynamic loading
Materials Science and Engineering C 32 (2012) 1548–1553
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Radiation sterilized bone response to dynamic loading Marcin Mardas a,⁎, Leszek Kubisz b, Piotr Biskupski c, Sławomir Mielcarek c, Marta Stelmach-Mardas d, Iwona Kałuska e a
Department of Oncology, Poznan University of Medical Sciences, ul. Szamarzewskiego 82/84, 60-569 Poznan, Poland Department of Biophysics, Poznan University of Medical Sciences, ul. Fredry 10, 61-701 Poznan, Poland Department of Physics, Adam Mickiewicz University, ul. Umultowska 85, 61-614 Poznan, Poland d Department of Bromatology, Poznan University of Medical Sciences, ul. Marcelińska 420, 60-354 Poznan, Poland e Centre for Radiation Research and Technology, Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 28 November 2011 Received in revised form 27 February 2012 Accepted 20 April 2012 Available online 28 April 2012 Keywords: DMA (Dynamic Mechanical Analysis) Bone Irradiation Denaturation temperature Activation energy
a b s t r a c t Allogeneic bone grafts are used on a large scale in surgeries. To avoid the risk of infectious diseases, allografts should be radiation-sterilized. So far, no international consensus has been achieved regarding the optimal radiation dose. Many authors suggest that bone sterilization deteriorates bone mechanical properties. However, no data on the influence of ionizing radiation on bone dynamic mechanical properties are available. Bovine femurs from 2-year old animal were machine cut and irradiated with the doses 10, 15, 25, 35, 45 and 50 kGy. Dynamic mechanical analysis was performed at 1–10 Hz at the temperature range of 0–350 °C in 3point bending configuration. No statistically significant differences in storage modulus were observed. However, there were significant decreased values of loss modulus between the samples irradiated with doses of 10 (↓14.3%), 15, 45 and 50 kGy (↓33.2%) and controls. It was stated that increased irradiation dose decreases the temperature where collagen denaturation process starts and increases the temperature where the collagen denaturation process finishes. It was shown that activation energy of denaturation process is significantly higher for the samples irradiated with the dose of 50 kGy (615 kJ/mol) in comparison with control samples and irradiation with other doses (100–135 kJ/mol). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Allogeneic bone grafts are used on a large scale in surgeries, from massive bone grafts to milled bones, where they are used as a filling material [1–4]. Approximately 15% of all the reconstructive surgeries require using bone grafts, the bigger the defect that requires reconstructing is, the greater the recommendations to use the allogeneic bones are [5]. Only in the United States 450 thousands allogeneic bone grafts are performed a year [6]. The number of donors is rising every year as well [7]. Widespread availability of allogeneic bones is undoubtedly considered an advantage. Apart from this, unlike allogeneic bone grafts, the extraction of autogeneic grafts involves patient's additional suffering and runs a higher risk of a surgery [8]. The risk of infectious disease transmission from donor to recipient limits the use of fresh frozen human tissue grafts. To avoid this risk, allografts should be radiation-sterilized [9]. In general, the application of irradiation for terminal sterilization of bone allograft is well accepted, but the dose of gamma radiation is still controversial [2]. So far, no international consensus has been achieved regarding the
⁎ Corresponding author. Tel./fax: + 48 618510490. E-mail address: [email protected] (M. Mardas). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.04.041
optimal radiation dose therefore the recommended doses range between 15 and 35 kGy [1]. Dose selection is a kind of compromise as it has to be high enough to inactivate all microorganisms and simultaneously low enough to maintain the mechanical properties of bones [10]. The sterilization efficiency of ionizing radiation lies in its good penetrability inside matter and its high effectiveness in the inactivation of pathogens [11]. Ionizing radiation affects the bone in two ways. The first direct way occurs in a dry state, when collagen polypeptide chains are damaged and torn. The second indirect mechanism is more dominating with water presence when due to ionizing radiation water radiolysis occurs and free oxygen radicals are formed. In this case, cross‐linking of collagen fibers takes place [1,11,12]. Many authors [13–18] suggest that bone sterilization with ionizing radiation deteriorates bone mechanical properties. However, all data concerning this issue and available in literature are based on static tests. Currently, apart from preliminary research [19], no other results on the influence of ionizing radiation on bone dynamic mechanical properties are available. However, most bone fractures occur under dynamic loading conditions, under which the viscoelasticity of bone may have a pronounced effect [20]. The difference between DMA (Dynamic Mechanical Analysis) and static research is that in DMA analysis, the body is subjected to oscillatory force which causes sinusoidal stress. The response to the sinusoidal stress is the sinusoidal strain, where stress and strain are
M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553
shifted in time [21,22]. If it is assumed that strain changes according to Eq. (1), the stress can be expressed with the following Eq. (2): ε ¼ ε0 ⋅sinðω⋅t Þ
ð1Þ
σ ¼ σ 0 ⋅sinðω⋅t þ δÞ
ð2Þ
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With the help of the software NETZSCH Peak Separation particular peaks were marked, which were identified as the beginning and the end of collagen denaturation temperature. The peaks were marked for the value E” at the frequency of 1 Hz due to the fact, that it is the closest to the strains existing in physiological conditions [23]. 3. Results
where: ε0 – strain amplitude, ω – angular frequency, δ – phase shift angle. Among the parameters measured in DMA research, the following modules are listed: storage modulus (E’), loss modulus (E”) and complex modulus (E*). The modules provide better characteristics of the studied material due to the possibility of measuring the capacity of stored energy (E’), as well as its distracting (E”) [21,22]. It seems that research involving dynamic method is to a great extent closer to physiological conditions than the research conducted with static method [23]. E ¼ E’ þ iE”
ð3Þ
The aim of the current study was to evaluate the influence of ionizing radiation on dynamic mechanical properties of a bone using dynamic mechanical analysis. 2. Materials and methods Bovine femurs from a 2-year-old animal, immediately after slaughter, have been used for the research. To minimize the influence of biological variability on the analyzed material, bones from one animal have been used. After mechanical cleaning to get rid of soft tissues, the middle part of the corpus femoris made of compact bone has been chosen for further treatment. Afterwards, cuboidal samples with the parameters 30 × 5 × 2 mm have been extracted from the bone shaft with a diamond saw especially constructed for this purpose. The longest parameter is parallel to long bone axis. During the cutting process, the bone was being cooled with water to avoid temperature increase and thermal denaturation of collagen. The samples have been ground to obtain smooth, flat parallel surface. During the grinding process the samples have also been cooled. After drying at room temperature each sample was packed separately in a disposable container made from PET foil. The samples were radiated with an electron beam of the energy 10 MeV with a given current of 550 mA. The samples were divided into 7 groups depending of the radiation dose: 0 (control), 10, 15, 25, 35, 45 and 50 kGy (4 samples in every group). DMA examination was conducted with DMA 242 analyzer made by Netzsch. The research: E ¼
The exemplary graph of the control sample of temperature measurement course E’ and E” was presented in Fig. 1. Originally, the values E’ were decreasing while the temperature was rising and after exceeding 50 °C they were successively rising. Having exceeded this temperature, the dispersion of E’ value was also noticed. While reaching 200 °C local maximum was met, after which there was a rapid decrease of the value E’. Similar changes were observed for the temperature measurement courses E”. After initial slight decrease of E” value, there was an increase and local maximum, afterwards the dispersion of E” value was noticed and their irregular course. Having exceeded 200 °C local maximum was also observed followed by E” value decrease. One more local maximum was noticed at the temperature of about 330– 350 °C. Additionally, it was found that in each case the frequency increase led to E’ value increase and E” value decrease. Figs. 2 and 3 present the average values with standard deviations of E’ and E”. No statistically significant differences of E’ values were found between the researched groups. In case of E” values, the differences were noticed for the samples irradiated with 10 kGy dose within the temperature range 25–50 °C and for the samples irradiated with 50 kGy dose within the temperature range 0–8 °C. The differences were also observed within the temperature range above 260 °C for the samples irradiated with the doses of 10, 15 and 45, 50 kGy. 4. Discussion Changes of E’ and E” values inflicted by heating showed similar character regardless of irradiation dose. Originally, the decrease of the values E’ and E” was noticed, followed by a change of a trend for the increasing one at the temperature range of 50–70 °C. Mano [23] obtained similar results examining the chicken bone at the temperature range of −50 °C–80 °C, however, the change of the trend was noticed after exceeding 0 °C. Further temperature increase led to dispersion of E’ and E” values, which started at the temperature of about 120 °C, which is most probably related with the beginning of collagen denaturation process. This process, depending on the way of sample preparation, collagen origin and research method, takes places at the temperature range of 100–200 °C [24]. Kronick and Cooke [25] in DSC (Differential Scanning Calorimetry) research of
3
l F ⋅ 4bh3 a
ð4Þ
where: E* – complex modulus [Pa], l – sample length [mm], b – wideness [mm], h – sample height [mm], F – strength [Pa], a* – total dynamic displacement [mm]. The pressure strength equals 1 N. The measurements were conducted at the temperature ranging from 0 °C to 350 °C, heating them with a fixed speed of 3 °C/min. Before the measurements, the samples were cooled in a liquid nitrogen environment. The measured parameters E’ and E” have been established for four frequencies: 1, 2, 5 and 10 Hz. 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 6.0 PL package. Friedman and Wilcoxon tests were applied for the comparisons between the groups and Spearman's Rank correlation coefficient (r), assuming the confidence level of α = 0.05.
Fig. 1. Values E’ and E” for the control sample.
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M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553
Fig. 2. The comparison of average E’ values ± SD between the groups irradiated with different doses.
bovine bone indicated the temperature of collagen denaturation at the value of approximately 155 °C. Wang et al. [26] while examining the human bone showed that denaturation of collagen starts after exceeding the temperature of 120 °C. They also underlined, that at the temperature of 160 °C the degree of collagen denaturation reached about 50%, and at 190 °C collagen was denaturated at 100%. Bowman et al. [27] proved in their research that denaturation temperature of decalcified bovine collagen equals 50–56 °C, which proves that there is a protective effect of lattice on collagen. Furthermore, it was proved that the temperature of collagen denaturation depends on the degree of hydratation and decreasing the amount of water leads to the denaturation temperature increase [28–31]. In the present research, the temperature where strong dispersion of E” value starts was taken as the measurement of the influence of
irradiation dose on the process of the beginning of denaturation. The obtained results were collected and presented in Fig. 4. It was stated that increased irradiation dose decreases the temperature where collagen denaturation process starts. It can be related to the fact that partially destroyed collagen fibers are more subject to denaturation. Ionizing radiation influences the bone in two ways. When the bone is dry the direct effect is more dominating, breaking the collagen fibers. When the bone is wet, the indirect effect is prevalent – collagen cross-linking; both processes can occur simultaneously [11]. The temperature of the peak appearing at 200 °C was assumed in this research as the temperature of the end of the collagen thermal denaturation process, according to Wang and Feng [20]. The temperature relation of this peak to the dose value was presented in Fig. 5. It was shown that when the irradiation dose is increased, the
M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553
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Fig. 3. The comparison of average E” values ± SD between the groups irradiated with different doses.
Fig. 4. Temperature of initiating the denaturation process depending on the irradiation dose.
temperature where the collagen denaturation process finishes increases as well. This phenomenon is related with the increasing process of collagen cross-linking while the dose is increased. It must be underlined that this phenomenon must not be linked with the degradation of the lattice as this phenomenon occurs after exceeding the temperature of 400–600 °C, according to different authors [20,32]. E’ value for the native bone, depending on its origin and experimental conditions range between 8 and 20 GPa [20,23,24,33–36]. It can be stated that indicated E’ values for the irradiated bone are compliant with those presented by other authors. When it comes to E” average values, they equaled approximately 900 MPa at room temperature. According to Wang and Feng [20], E” value, also for bovine bone, equaled approximately 220 MPa at room temperature. Due to the fact that loss modulus (E”) is the measurement of bone energy distraction during cyclic strains, the knowledge of its value
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M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553
Fig. 5. Temperature of ending the denaturation process depending on the irradiation dose.
helps to determine the bone ability to resist the pressure strength until the bone is broken. Decreased E” value means in practice an increased risk of bone breaking [20]. In the conducted experiment, significant statistic differences for the samples irradiated with the dose of 10, 15, 45 and 50 kGy were shown. In the case of 10 kGy it was the reduction by 14.3%, in the case of 50 kGy it reached as much as 33.2%. Differences between samples irradiated with the dose 15 and 45 kGy were only at high temperatures observed. Storage modulus (E’) can on the other hand be considered the equivalent of Young modulus in DMA [34]. No significant statistical differences for E’ value were found during the conducted experiment. The highest difference (↓13.7%) was observed for the samples irradiated with the dose of 50 kGy (p > 0.05). The obtained results are concurrent with the results of other authors. Currey [14] in his static research did not show any changes in Young modulus for the samples irradiated with the doses of 17, 29.5 and as high as 94.7 kGy, despite the fact that changes were observed in other parameters. Similarly, Balsly et al. [37] and Zhang [38] did not show changes in Young modulus for the bones irradiated with the doses of 18–25 kGy. Moreover, Kamieński et al. [9] did not obtain significant statistical extension resistance differences between the samples irradiated with the doses of 25, 35, 50, and even 100 kGy. Nevertheless, it is suggested that ionizing radiation increases the bone stiffness, which was confirmed in the research of Butler et al. [39]. Subsequently, using the Arrhenius equation, which combines the relaxation frequency with activation energy and temperature, where the relation occurs, the activation energy of the denaturation process was established and the results are presented in Fig. 6. lnυr ¼ lnυ0 −
Ea 1 ⋅ k T
ð5Þ
Where: vr – relaxation frequency, v0 – frequency of thermal vibrations, Ea – relaxation process activation energy (J/molecule), k – Boltzmann constant, T – temperature. It was shown that activation energy of denaturation process is significantly higher for the samples irradiated with the dose of 50 kGy (615 kJ/mol) in comparison with control samples and the samples irradiated with other doses (p b 0,001). Schaller et al. [35] in their DMA research achieved the activation energy of approximately 130 kJ/mol, thus similar to those observed for control samples (120 kJ/mol) and irradiated with the dose to 45 kGy in this research. Higher values of activation energy (320–360 kJ/mol), were observed also for the native bone while conducting DSC research [40,41]. Literature contains no information concerning activation energy of denaturation process for irradiated samples. Such high activation energy of denaturation process for the sample irradiated with the dose of 50 kGy probably results from significant collagen crosslinking.
Fig. 6. Activation energy of collagen denaturation process depending on the dose.
5. Conclusions The research showed that ionizing irradiation deteriorates the dynamic mechanical properties of bones. Although no relevant statistical differences of the storage modulus value of the irradiated bones were shown, the differences in the case of loss modulus were noticed – the highest when the bones were irradiated with the dose of 50 kGy. Additionally, it was shown that ionizing irradiation influences the process of collagen thermal denaturation, decreasing the temperature at which the process is initiated and increasing the temperature at which it is terminated. It was also shown that the process activation energy of collagen denaturation is significantly higher for the samples irradiated with the dose of 50 kGy in comparison with other samples. As far as the dynamic mechanical properties of bones are concerned, the 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.
References [1] H. Nguyen, D.A. Morgan, M.R. Forwood, Cell Tissue Bank. 8 (2) (2007) 93–105. [2] H. Nguyen, D.A. Morgan, M.R. Forwood, Cell Tissue Bank. 8 (2) (2007) 81–91. [3] T.N. Board, S. Brunskill, C. Doree, C. Hyde, P.R. Kay, R.D. Meek, R. Webster, G. Galea, Cochrane Database Syst. Rev. (4) (2009) (Art. No.: CD006351). [4] R. Kohler, F. Lorge, M. Brunat-Mentigny, D. Noyer, L. Patricot, Int. Orthop. 14 (1990) 249–253. [5] G.O. Hofmann, M.H. Kirschner, T. Wangemann, C. Falk, W. Mempel, C. Hammer, Arch. Orthop. Trauma Surg. 114 (1995) 159–166. [6] O. Akkus, R.M. Belaney, J. Orthop. Res. 23 (5) (2005) 1054–1058. [7] C.N. Cornell, Bull. Hosp. Jt. Dis. 62 (1–2) (2004) 13–17. [8] M.G. Andrade, C.N. Sá, A.M. Marchionni, T.C. dos Santos Calmon de Bittencourt, M. Sadigursky, Cell Tissue Bank. 9 (4) (2008) 279–287. [9] A. Kamiński, G. Gut, J. Marowska, M. Lada-Kozłowska, W. Biwejnis, M. Zasacka, Cell Tissue Bank. 10 (3) (2009) 215–219. [10] L. Kubisz, W: Sickafus KE (red.), Radiation effects in solids, Springer, 2007, pp. 575–588. [11] A. Dziedzic-Goclawska, A. Kaminski, I. Uhrynowska-Tyszkiewicz, W. Stachowicz, Cell Tissue Bank. 6 (3) (2005) 201–219. [12] O. Akkus, R.M. Belaney, P. Das, J. Orthop. Res. 23 (4) (Jul. 2005) 838–845. [13] P. Hernigou, G. Gras, G. Marinello, D. Dormont, Acta Orthop. Scand. 71 (5) (2000) 508–512. [14] J. Currey, J. Foreman, I. Laketic, J. Mitchell, D.E. Pegg, G.C. Reilly, J. Orthop. Res. 15 (1) (1997) 111–117. [15] A. Komender, Mater. Med. Pol. 8 (1976) 13. [16] N. Triantafyllou, E. Sotiropoulos, J.N. Triantafyllou, Acta Orthop. Belg. 41 (Suppl. 1(1)) (1975) 35–44. [17] A.J. Hamer, J.R. Strachan, M.M. Black, C.J. Ibbotson, I. Stockley, R.A. Elson, J. Bone Joint Surg. Br. 78 (3) (1996) 363–368. [18] O. Akkus, C.M. Rimnac, J. Orthop. Res. 19 (2001) 927–934. [19] L. Kubisz, S. Mielcarek, P. Biskupski, M. Mardas, Pol. J. Environ. Stud. 15 (4a) (2006) 72–74. [20] T. Wang, Z. Feng, Mater. Lett. 59 (2005) 2277–2280. [21] H.P. Menard, Dynamic Mechanical Analysis: A practical introduction, second edition CRC Press, 2008. [22] D.S. Jones, Int. J. Pharm. 179 (1999) 167–178. [23] J.F. Mano, Mater. Sci. Eng. C 25 (2005) 145–152.
M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553 [24] J. Yamashita, X. Li, B.R. Furman, H.R. Rawls, X. Wang, C.M. Agrawal, J. Biomed. Mater. Res. 63 (2002) 31–36. [25] P.L. Kronick, P. Cooke, Connect. Tissue Res. 33 (4) (1996) 275–282. [26] X. Wang, R.A. Bank, J.M. TeKoppele, C.M. Agrawal, J. Orthop. Res. 19 (6) (Nov. 2001) 1021–1026. [27] S.M. Bowman, L.J. Gibson, W.C. Hayes, T.A. McMahon, J. Biomech. Eng. 121 (2) (1999) 253–258. [28] C. Miles, M. Ghelashvili, Biophys. J. 76 (6) (1999) 3243–3252. [29] A.L. Nguyen, B.T. Vu, G.L. Wilkes, Biopolymers 13 (1974) 1023–1037. [30] C. Miles, A. Bailey, Proc. Indian Acad. Sci. 111 (1) (1999) 71–80. [31] C.A. Miles, N.C. Avery, V.V. Rodin, A.J. Bailey, J. Mol. Biol. 346 (2005) 551–556. [32] F. Peters, K. Schwarz, M. Epple, Thermochim. Acta 361 (2000) 131–138. [33] X.N. Dong, Y.N. Yeni, C.M. Les, D.P. Fyhrie, J. Biomed. Mater. Res. A 68 (3) (2004) 573–583.
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[34] Y.N. Yeni, G.T. Christopherson, A.S. Turner, C.M. Les, D.P. Fyhrie, J. Biomed. Mater. Res. A 69 (1) (2004) 124–130. [35] R. Schaller, S. Barrault, P. Zysset, Mater. Sci. Eng. A 370 (2004) 569–574. [36] J. Yamashita, B.R. Furman, H.R. Rawls, X. Wang, C.M. Agrawal, J. Biomed. Mater. Res. 58 (1) (2001) 47–53. [37] C.R. Balsly, A.T. Cotter, L.A. Williams, B.D. Gaskins, M.A. Moore, L. Wolfinbarger Jr., Cell Tissue Bank. 9 (4) (2008) 289–298. [38] Y. Zhang, D. Homsi, K. Gates, K. Oakes, V. Sutherland, L. Wolfinbarger Jr., Spine 19 (3) (1994) 304–308. [39] A.M. Butler, D.A. Morgan, R. Verheul, W.R. Walsh, Biomaterials 26 (30) (2005) 6009–6013. [40] H. Trębacz, K. Wójtowicz, Int. J. Biol. Macromol. 37 (5) (2005) 257–262. [41] S. Vyazovkin, L. Vincent, N. Sbirrazzuoli, Macromol. Biosci. 7 (11) (2007) 1181–1186.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Radiation sterilized bone response to dynamic loading Marcin Mardas a,⁎, Leszek Kubisz b, Piotr Biskupski c, Sławomir Mielcarek c, Marta Stelmach-Mardas d, Iwona Kałuska e a
Department of Oncology, Poznan University of Medical Sciences, ul. Szamarzewskiego 82/84, 60-569 Poznan, Poland Department of Biophysics, Poznan University of Medical Sciences, ul. Fredry 10, 61-701 Poznan, Poland Department of Physics, Adam Mickiewicz University, ul. Umultowska 85, 61-614 Poznan, Poland d Department of Bromatology, Poznan University of Medical Sciences, ul. Marcelińska 420, 60-354 Poznan, Poland e Centre for Radiation Research and Technology, Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 28 November 2011 Received in revised form 27 February 2012 Accepted 20 April 2012 Available online 28 April 2012 Keywords: DMA (Dynamic Mechanical Analysis) Bone Irradiation Denaturation temperature Activation energy
a b s t r a c t Allogeneic bone grafts are used on a large scale in surgeries. To avoid the risk of infectious diseases, allografts should be radiation-sterilized. So far, no international consensus has been achieved regarding the optimal radiation dose. Many authors suggest that bone sterilization deteriorates bone mechanical properties. However, no data on the influence of ionizing radiation on bone dynamic mechanical properties are available. Bovine femurs from 2-year old animal were machine cut and irradiated with the doses 10, 15, 25, 35, 45 and 50 kGy. Dynamic mechanical analysis was performed at 1–10 Hz at the temperature range of 0–350 °C in 3point bending configuration. No statistically significant differences in storage modulus were observed. However, there were significant decreased values of loss modulus between the samples irradiated with doses of 10 (↓14.3%), 15, 45 and 50 kGy (↓33.2%) and controls. It was stated that increased irradiation dose decreases the temperature where collagen denaturation process starts and increases the temperature where the collagen denaturation process finishes. It was shown that activation energy of denaturation process is significantly higher for the samples irradiated with the dose of 50 kGy (615 kJ/mol) in comparison with control samples and irradiation with other doses (100–135 kJ/mol). © 2012 Elsevier B.V. All rights reserved.
1. Introduction Allogeneic bone grafts are used on a large scale in surgeries, from massive bone grafts to milled bones, where they are used as a filling material [1–4]. Approximately 15% of all the reconstructive surgeries require using bone grafts, the bigger the defect that requires reconstructing is, the greater the recommendations to use the allogeneic bones are [5]. Only in the United States 450 thousands allogeneic bone grafts are performed a year [6]. The number of donors is rising every year as well [7]. Widespread availability of allogeneic bones is undoubtedly considered an advantage. Apart from this, unlike allogeneic bone grafts, the extraction of autogeneic grafts involves patient's additional suffering and runs a higher risk of a surgery [8]. The risk of infectious disease transmission from donor to recipient limits the use of fresh frozen human tissue grafts. To avoid this risk, allografts should be radiation-sterilized [9]. In general, the application of irradiation for terminal sterilization of bone allograft is well accepted, but the dose of gamma radiation is still controversial [2]. So far, no international consensus has been achieved regarding the
⁎ Corresponding author. Tel./fax: + 48 618510490. E-mail address: [email protected] (M. Mardas). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.04.041
optimal radiation dose therefore the recommended doses range between 15 and 35 kGy [1]. Dose selection is a kind of compromise as it has to be high enough to inactivate all microorganisms and simultaneously low enough to maintain the mechanical properties of bones [10]. The sterilization efficiency of ionizing radiation lies in its good penetrability inside matter and its high effectiveness in the inactivation of pathogens [11]. Ionizing radiation affects the bone in two ways. The first direct way occurs in a dry state, when collagen polypeptide chains are damaged and torn. The second indirect mechanism is more dominating with water presence when due to ionizing radiation water radiolysis occurs and free oxygen radicals are formed. In this case, cross‐linking of collagen fibers takes place [1,11,12]. Many authors [13–18] suggest that bone sterilization with ionizing radiation deteriorates bone mechanical properties. However, all data concerning this issue and available in literature are based on static tests. Currently, apart from preliminary research [19], no other results on the influence of ionizing radiation on bone dynamic mechanical properties are available. However, most bone fractures occur under dynamic loading conditions, under which the viscoelasticity of bone may have a pronounced effect [20]. The difference between DMA (Dynamic Mechanical Analysis) and static research is that in DMA analysis, the body is subjected to oscillatory force which causes sinusoidal stress. The response to the sinusoidal stress is the sinusoidal strain, where stress and strain are
M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553
shifted in time [21,22]. If it is assumed that strain changes according to Eq. (1), the stress can be expressed with the following Eq. (2): ε ¼ ε0 ⋅sinðω⋅t Þ
ð1Þ
σ ¼ σ 0 ⋅sinðω⋅t þ δÞ
ð2Þ
1549
With the help of the software NETZSCH Peak Separation particular peaks were marked, which were identified as the beginning and the end of collagen denaturation temperature. The peaks were marked for the value E” at the frequency of 1 Hz due to the fact, that it is the closest to the strains existing in physiological conditions [23]. 3. Results
where: ε0 – strain amplitude, ω – angular frequency, δ – phase shift angle. Among the parameters measured in DMA research, the following modules are listed: storage modulus (E’), loss modulus (E”) and complex modulus (E*). The modules provide better characteristics of the studied material due to the possibility of measuring the capacity of stored energy (E’), as well as its distracting (E”) [21,22]. It seems that research involving dynamic method is to a great extent closer to physiological conditions than the research conducted with static method [23]. E ¼ E’ þ iE”
ð3Þ
The aim of the current study was to evaluate the influence of ionizing radiation on dynamic mechanical properties of a bone using dynamic mechanical analysis. 2. Materials and methods Bovine femurs from a 2-year-old animal, immediately after slaughter, have been used for the research. To minimize the influence of biological variability on the analyzed material, bones from one animal have been used. After mechanical cleaning to get rid of soft tissues, the middle part of the corpus femoris made of compact bone has been chosen for further treatment. Afterwards, cuboidal samples with the parameters 30 × 5 × 2 mm have been extracted from the bone shaft with a diamond saw especially constructed for this purpose. The longest parameter is parallel to long bone axis. During the cutting process, the bone was being cooled with water to avoid temperature increase and thermal denaturation of collagen. The samples have been ground to obtain smooth, flat parallel surface. During the grinding process the samples have also been cooled. After drying at room temperature each sample was packed separately in a disposable container made from PET foil. The samples were radiated with an electron beam of the energy 10 MeV with a given current of 550 mA. The samples were divided into 7 groups depending of the radiation dose: 0 (control), 10, 15, 25, 35, 45 and 50 kGy (4 samples in every group). DMA examination was conducted with DMA 242 analyzer made by Netzsch. The research: E ¼
The exemplary graph of the control sample of temperature measurement course E’ and E” was presented in Fig. 1. Originally, the values E’ were decreasing while the temperature was rising and after exceeding 50 °C they were successively rising. Having exceeded this temperature, the dispersion of E’ value was also noticed. While reaching 200 °C local maximum was met, after which there was a rapid decrease of the value E’. Similar changes were observed for the temperature measurement courses E”. After initial slight decrease of E” value, there was an increase and local maximum, afterwards the dispersion of E” value was noticed and their irregular course. Having exceeded 200 °C local maximum was also observed followed by E” value decrease. One more local maximum was noticed at the temperature of about 330– 350 °C. Additionally, it was found that in each case the frequency increase led to E’ value increase and E” value decrease. Figs. 2 and 3 present the average values with standard deviations of E’ and E”. No statistically significant differences of E’ values were found between the researched groups. In case of E” values, the differences were noticed for the samples irradiated with 10 kGy dose within the temperature range 25–50 °C and for the samples irradiated with 50 kGy dose within the temperature range 0–8 °C. The differences were also observed within the temperature range above 260 °C for the samples irradiated with the doses of 10, 15 and 45, 50 kGy. 4. Discussion Changes of E’ and E” values inflicted by heating showed similar character regardless of irradiation dose. Originally, the decrease of the values E’ and E” was noticed, followed by a change of a trend for the increasing one at the temperature range of 50–70 °C. Mano [23] obtained similar results examining the chicken bone at the temperature range of −50 °C–80 °C, however, the change of the trend was noticed after exceeding 0 °C. Further temperature increase led to dispersion of E’ and E” values, which started at the temperature of about 120 °C, which is most probably related with the beginning of collagen denaturation process. This process, depending on the way of sample preparation, collagen origin and research method, takes places at the temperature range of 100–200 °C [24]. Kronick and Cooke [25] in DSC (Differential Scanning Calorimetry) research of
3
l F ⋅ 4bh3 a
ð4Þ
where: E* – complex modulus [Pa], l – sample length [mm], b – wideness [mm], h – sample height [mm], F – strength [Pa], a* – total dynamic displacement [mm]. The pressure strength equals 1 N. The measurements were conducted at the temperature ranging from 0 °C to 350 °C, heating them with a fixed speed of 3 °C/min. Before the measurements, the samples were cooled in a liquid nitrogen environment. The measured parameters E’ and E” have been established for four frequencies: 1, 2, 5 and 10 Hz. 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 6.0 PL package. Friedman and Wilcoxon tests were applied for the comparisons between the groups and Spearman's Rank correlation coefficient (r), assuming the confidence level of α = 0.05.
Fig. 1. Values E’ and E” for the control sample.
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Fig. 2. The comparison of average E’ values ± SD between the groups irradiated with different doses.
bovine bone indicated the temperature of collagen denaturation at the value of approximately 155 °C. Wang et al. [26] while examining the human bone showed that denaturation of collagen starts after exceeding the temperature of 120 °C. They also underlined, that at the temperature of 160 °C the degree of collagen denaturation reached about 50%, and at 190 °C collagen was denaturated at 100%. Bowman et al. [27] proved in their research that denaturation temperature of decalcified bovine collagen equals 50–56 °C, which proves that there is a protective effect of lattice on collagen. Furthermore, it was proved that the temperature of collagen denaturation depends on the degree of hydratation and decreasing the amount of water leads to the denaturation temperature increase [28–31]. In the present research, the temperature where strong dispersion of E” value starts was taken as the measurement of the influence of
irradiation dose on the process of the beginning of denaturation. The obtained results were collected and presented in Fig. 4. It was stated that increased irradiation dose decreases the temperature where collagen denaturation process starts. It can be related to the fact that partially destroyed collagen fibers are more subject to denaturation. Ionizing radiation influences the bone in two ways. When the bone is dry the direct effect is more dominating, breaking the collagen fibers. When the bone is wet, the indirect effect is prevalent – collagen cross-linking; both processes can occur simultaneously [11]. The temperature of the peak appearing at 200 °C was assumed in this research as the temperature of the end of the collagen thermal denaturation process, according to Wang and Feng [20]. The temperature relation of this peak to the dose value was presented in Fig. 5. It was shown that when the irradiation dose is increased, the
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Fig. 3. The comparison of average E” values ± SD between the groups irradiated with different doses.
Fig. 4. Temperature of initiating the denaturation process depending on the irradiation dose.
temperature where the collagen denaturation process finishes increases as well. This phenomenon is related with the increasing process of collagen cross-linking while the dose is increased. It must be underlined that this phenomenon must not be linked with the degradation of the lattice as this phenomenon occurs after exceeding the temperature of 400–600 °C, according to different authors [20,32]. E’ value for the native bone, depending on its origin and experimental conditions range between 8 and 20 GPa [20,23,24,33–36]. It can be stated that indicated E’ values for the irradiated bone are compliant with those presented by other authors. When it comes to E” average values, they equaled approximately 900 MPa at room temperature. According to Wang and Feng [20], E” value, also for bovine bone, equaled approximately 220 MPa at room temperature. Due to the fact that loss modulus (E”) is the measurement of bone energy distraction during cyclic strains, the knowledge of its value
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Fig. 5. Temperature of ending the denaturation process depending on the irradiation dose.
helps to determine the bone ability to resist the pressure strength until the bone is broken. Decreased E” value means in practice an increased risk of bone breaking [20]. In the conducted experiment, significant statistic differences for the samples irradiated with the dose of 10, 15, 45 and 50 kGy were shown. In the case of 10 kGy it was the reduction by 14.3%, in the case of 50 kGy it reached as much as 33.2%. Differences between samples irradiated with the dose 15 and 45 kGy were only at high temperatures observed. Storage modulus (E’) can on the other hand be considered the equivalent of Young modulus in DMA [34]. No significant statistical differences for E’ value were found during the conducted experiment. The highest difference (↓13.7%) was observed for the samples irradiated with the dose of 50 kGy (p > 0.05). The obtained results are concurrent with the results of other authors. Currey [14] in his static research did not show any changes in Young modulus for the samples irradiated with the doses of 17, 29.5 and as high as 94.7 kGy, despite the fact that changes were observed in other parameters. Similarly, Balsly et al. [37] and Zhang [38] did not show changes in Young modulus for the bones irradiated with the doses of 18–25 kGy. Moreover, Kamieński et al. [9] did not obtain significant statistical extension resistance differences between the samples irradiated with the doses of 25, 35, 50, and even 100 kGy. Nevertheless, it is suggested that ionizing radiation increases the bone stiffness, which was confirmed in the research of Butler et al. [39]. Subsequently, using the Arrhenius equation, which combines the relaxation frequency with activation energy and temperature, where the relation occurs, the activation energy of the denaturation process was established and the results are presented in Fig. 6. lnυr ¼ lnυ0 −
Ea 1 ⋅ k T
ð5Þ
Where: vr – relaxation frequency, v0 – frequency of thermal vibrations, Ea – relaxation process activation energy (J/molecule), k – Boltzmann constant, T – temperature. It was shown that activation energy of denaturation process is significantly higher for the samples irradiated with the dose of 50 kGy (615 kJ/mol) in comparison with control samples and the samples irradiated with other doses (p b 0,001). Schaller et al. [35] in their DMA research achieved the activation energy of approximately 130 kJ/mol, thus similar to those observed for control samples (120 kJ/mol) and irradiated with the dose to 45 kGy in this research. Higher values of activation energy (320–360 kJ/mol), were observed also for the native bone while conducting DSC research [40,41]. Literature contains no information concerning activation energy of denaturation process for irradiated samples. Such high activation energy of denaturation process for the sample irradiated with the dose of 50 kGy probably results from significant collagen crosslinking.
Fig. 6. Activation energy of collagen denaturation process depending on the dose.
5. Conclusions The research showed that ionizing irradiation deteriorates the dynamic mechanical properties of bones. Although no relevant statistical differences of the storage modulus value of the irradiated bones were shown, the differences in the case of loss modulus were noticed – the highest when the bones were irradiated with the dose of 50 kGy. Additionally, it was shown that ionizing irradiation influences the process of collagen thermal denaturation, decreasing the temperature at which the process is initiated and increasing the temperature at which it is terminated. It was also shown that the process activation energy of collagen denaturation is significantly higher for the samples irradiated with the dose of 50 kGy in comparison with other samples. As far as the dynamic mechanical properties of bones are concerned, the 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.
References [1] H. Nguyen, D.A. Morgan, M.R. Forwood, Cell Tissue Bank. 8 (2) (2007) 93–105. [2] H. Nguyen, D.A. Morgan, M.R. Forwood, Cell Tissue Bank. 8 (2) (2007) 81–91. [3] T.N. Board, S. Brunskill, C. Doree, C. Hyde, P.R. Kay, R.D. Meek, R. Webster, G. Galea, Cochrane Database Syst. Rev. (4) (2009) (Art. No.: CD006351). [4] R. Kohler, F. Lorge, M. Brunat-Mentigny, D. Noyer, L. Patricot, Int. Orthop. 14 (1990) 249–253. [5] G.O. Hofmann, M.H. Kirschner, T. Wangemann, C. Falk, W. Mempel, C. Hammer, Arch. Orthop. Trauma Surg. 114 (1995) 159–166. [6] O. Akkus, R.M. Belaney, J. Orthop. Res. 23 (5) (2005) 1054–1058. [7] C.N. Cornell, Bull. Hosp. Jt. Dis. 62 (1–2) (2004) 13–17. [8] M.G. Andrade, C.N. Sá, A.M. Marchionni, T.C. dos Santos Calmon de Bittencourt, M. Sadigursky, Cell Tissue Bank. 9 (4) (2008) 279–287. [9] A. Kamiński, G. Gut, J. Marowska, M. Lada-Kozłowska, W. Biwejnis, M. Zasacka, Cell Tissue Bank. 10 (3) (2009) 215–219. [10] L. Kubisz, W: Sickafus KE (red.), Radiation effects in solids, Springer, 2007, pp. 575–588. [11] A. Dziedzic-Goclawska, A. Kaminski, I. Uhrynowska-Tyszkiewicz, W. Stachowicz, Cell Tissue Bank. 6 (3) (2005) 201–219. [12] O. Akkus, R.M. Belaney, P. Das, J. Orthop. Res. 23 (4) (Jul. 2005) 838–845. [13] P. Hernigou, G. Gras, G. Marinello, D. Dormont, Acta Orthop. Scand. 71 (5) (2000) 508–512. [14] J. Currey, J. Foreman, I. Laketic, J. Mitchell, D.E. Pegg, G.C. Reilly, J. Orthop. Res. 15 (1) (1997) 111–117. [15] A. Komender, Mater. Med. Pol. 8 (1976) 13. [16] N. Triantafyllou, E. Sotiropoulos, J.N. Triantafyllou, Acta Orthop. Belg. 41 (Suppl. 1(1)) (1975) 35–44. [17] A.J. Hamer, J.R. Strachan, M.M. Black, C.J. Ibbotson, I. Stockley, R.A. Elson, J. Bone Joint Surg. Br. 78 (3) (1996) 363–368. [18] O. Akkus, C.M. Rimnac, J. Orthop. Res. 19 (2001) 927–934. [19] L. Kubisz, S. Mielcarek, P. Biskupski, M. Mardas, Pol. J. Environ. Stud. 15 (4a) (2006) 72–74. [20] T. Wang, Z. Feng, Mater. Lett. 59 (2005) 2277–2280. [21] H.P. Menard, Dynamic Mechanical Analysis: A practical introduction, second edition CRC Press, 2008. [22] D.S. Jones, Int. J. Pharm. 179 (1999) 167–178. [23] J.F. Mano, Mater. Sci. Eng. C 25 (2005) 145–152.
M. Mardas et al. / Materials Science and Engineering C 32 (2012) 1548–1553 [24] J. Yamashita, X. Li, B.R. Furman, H.R. Rawls, X. Wang, C.M. Agrawal, J. Biomed. Mater. Res. 63 (2002) 31–36. [25] P.L. Kronick, P. Cooke, Connect. Tissue Res. 33 (4) (1996) 275–282. [26] X. Wang, R.A. Bank, J.M. TeKoppele, C.M. Agrawal, J. Orthop. Res. 19 (6) (Nov. 2001) 1021–1026. [27] S.M. Bowman, L.J. Gibson, W.C. Hayes, T.A. McMahon, J. Biomech. Eng. 121 (2) (1999) 253–258. [28] C. Miles, M. Ghelashvili, Biophys. J. 76 (6) (1999) 3243–3252. [29] A.L. Nguyen, B.T. Vu, G.L. Wilkes, Biopolymers 13 (1974) 1023–1037. [30] C. Miles, A. Bailey, Proc. Indian Acad. Sci. 111 (1) (1999) 71–80. [31] C.A. Miles, N.C. Avery, V.V. Rodin, A.J. Bailey, J. Mol. Biol. 346 (2005) 551–556. [32] F. Peters, K. Schwarz, M. Epple, Thermochim. Acta 361 (2000) 131–138. [33] X.N. Dong, Y.N. Yeni, C.M. Les, D.P. Fyhrie, J. Biomed. Mater. Res. A 68 (3) (2004) 573–583.
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[34] Y.N. Yeni, G.T. Christopherson, A.S. Turner, C.M. Les, D.P. Fyhrie, J. Biomed. Mater. Res. A 69 (1) (2004) 124–130. [35] R. Schaller, S. Barrault, P. Zysset, Mater. Sci. Eng. A 370 (2004) 569–574. [36] J. Yamashita, B.R. Furman, H.R. Rawls, X. Wang, C.M. Agrawal, J. Biomed. Mater. Res. 58 (1) (2001) 47–53. [37] C.R. Balsly, A.T. Cotter, L.A. Williams, B.D. Gaskins, M.A. Moore, L. Wolfinbarger Jr., Cell Tissue Bank. 9 (4) (2008) 289–298. [38] Y. Zhang, D. Homsi, K. Gates, K. Oakes, V. Sutherland, L. Wolfinbarger Jr., Spine 19 (3) (1994) 304–308. [39] A.M. Butler, D.A. Morgan, R. Verheul, W.R. Walsh, Biomaterials 26 (30) (2005) 6009–6013. [40] H. Trębacz, K. Wójtowicz, Int. J. Biol. Macromol. 37 (5) (2005) 257–262. [41] S. Vyazovkin, L. Vincent, N. Sbirrazzuoli, Macromol. Biosci. 7 (11) (2007) 1181–1186.