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Effects of the freezing and thawing process on biomechanical properties of the human skull
Legal Medicine 16 (2014) 102–105
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Effects of the freezing and thawing process on biomechanical properties of the human skull Suguru Torimitsu a,⇑, Yoshifumi Nishida b, Tachio Takano b, Yoshinori Koizumi b, Mutsumi Hayakawa a, Daisuke Yajima a, Go Inokuchi a, Yohsuke Makino a, Ayumi Motomura a, Fumiko Chiba a, Hirotaro Iwase a a
Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Designing Everyday Life Function and Social System Team, Digital Human Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan b
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 25 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Cranial bones Frozen preservation Fracture load Stiffness
a b s t r a c t The aim of this study was to determine if biomechanical investigations of skull samples are reliable after skulls have been subjected to a freezing and thawing process. The skulls were obtained from 105 Japanese cadavers (66 males, 39 females) of known age that were autopsied in our department between October 2012 and June 2013. We obtained bone specimens from eight sites (four bilaterally symmetrical pairs) of each skull and measured the mass of each specimen. They were then classified into three groups (A, B, C) based on the duration of freezing of the experimental samples. The left-side samples were subjected to frozen storage (experimental group). The corresponding right-side samples were their controls. Bending tests were performed on the controls immediately after they were obtained. The experimental samples were preserved by refrigeration at 20 °C for 1 day (group A), 1 month (group B), or 3 months (group C). Following refrigeration, these samples were placed at 37 °C to thaw for 1 h and then were subjected to bending tests using a three-point-bending apparatus attached to a Handy force gauge. The device recorded the fracture load automatically when the specimen fractured. Statistical analyses revealed that there were no significant differences in sample fracture loads between the frozen preserved/thawed samples and the unfrozen controls for each of the cryopreservation intervals. We eliminated any possible sample mass bias by using controls from the same skull in each case. The results suggest that the freezing/thawing process has little effect on the mechanical properties of human skulls. Thus, frozen storage for up to 3 months is a good method for preserving human skulls. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction For forensic analysis of cases involving a fractured skull, it is important to estimate the applied external force. This is because skull fracture is an independent risk factor for intracranial lifethreatening complications such as epidural hematoma, subdural hematoma, and cerebral hemorrhagic contusion [1]. It is also vital to have a sound understanding of the biomechanical properties of the skull so that accurate estimates can be provided. Previous engineering studies have assessed the mechanical properties of human cranial bones using a variety of methods, including compression, tension, and bending tests [2–10]. During the last four decades, biomechanical studies have been conducted in forensic research as well [11]. The importance of such research requires that the skull be preserved in the best possible manner to avoid adding complications to the already difficult determinations.
⇑ Corresponding author. Tel.: +81 43 226 2078; fax: +81 43 226 2079. E-mail address: [email protected] (S. Torimitsu). 1344-6223/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2013.11.005
Some researchers have reported that for human long bones the freezing and thawing process does not significantly alter mechanical properties [12–18]. Hence, fresh human cadaver bones are usually preserved by refrigeration at 20 °C [12]. However, the effects of the freezing and thawing process on mechanical properties of human skull have not been previously studied. The aim of this study was to determine if biomechanical investigations are reliable after freezing and thawing skull samples.
2. Materials and methods 2.1. Subjects The skulls used in this research were obtained from 105 Japanese cadavers of known age and sex that were autopsied at the Department of Legal Medicine, Chiba University, between October 2012 and June 2013. The estimated postmortem interval of each body was within 7 days. Cases were excluded if the history highlighted conditions or events that could have affected the skull.
S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
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For example, skulls with a fracture, burns, obvious injury, or acquired or congenital abnormality were excluded from this study. The ethics committee of Chiba University approved the study. The skull samples were classified into three groups (A, B, C) as presented in Table 1. 2.2. Sampling During autopsy, bone specimens were obtained from eight cranial sites (four bilaterally symmetrical pairs) from each cadaver skull (left frontal, right frontal, left medial parietal, right medial parietal, left superolateral parietal, right superolateral parietal, left inferolateral parietal, and right inferolateral parietal bones). Fig 1 shows the orientations of samples extracted from each of the cadaver skulls. The length of each specimen was fixed at 50 mm and the width at 10 mm. Left and right frontal specimens were tangential to the left and right side of the coronal suture and along the anterior extended line of the sagittal suture, respectively. Left and right medial parietal specimens were tangential to the left and right side of the coronal suture and along the sagittal suture, respectively. Left and right superolateral and inferolateral parietal specimens were obtained from the lateral portion of the parietal bone immediately superior and inferior to the left and right superior temporal line, respectively. Samples were obtained using an oscillating saw and washed with saline. The mass (grams) of each sample was measured using an electronic scale that measured from 0.1 g to 600 g. A total of 840 samples (420 left-side and 420 right-side samples) were collected. Each of the three groups (A, B, C) comprised 280 samples (140 left-side and 140 right-side samples). The left-side (experimental) samples were subjected to frozen storage. The corresponding rightside samples were their controls in each case. The biomechanical test was performed on the control samples immediately after they were obtained. In contrast, the experimental samples were preserved in a biomedical freezer (MDF-U443; Sanyo, Osaka, Japan) with automatic defrosting function at 20 °C for 1 day (group A), 1 month (group B), or 3 months (group C). Following refrigeration, samples were placed at 37 °C to thaw for 1 h and then underwent biomechanical testing. 2.3. Biomechanical tests Bending tests, which have been used to determine the mechanical properties of various bones including human skull in previous studies [5,19,20], were performed on the skull samples using a three-point-bending apparatus (JSV-H1000; JISC, Nara, Japan) attached to a Handy force gauge (HF-100; JISC) as shown in Fig. 2.
Fig. 1. Orientations of samples extracted from the cadavers skulls.
Two lower supports were set 40 mm apart, and the specimen being tested was placed on the two supports. Above this setup was a 1000-N load cell that applied pressure at the center of the specimen from the outer surface at a constant speed of 100 lm/s. When the specimen fractured, the device automatically recorded the fracture load (N). 2.4. Statistical analysis The fracture load-to-sample mass ratio (FL/SM), in Newtons per gram, was determined by dividing the fracture load by the sample mass. Mann–Whitney U-tests were used to compare the fracture load, mass, and FL/SM between the experimental and control samples by site in each group. Differences of P < 0.05 were considered statistically significant. All statistical analyses were performed on a personal computer using Statistical Package for the Social Sciences (SPSS) version 21.0 software (IBM, Armonk, NY, USA). 3. Results and discussion Table 2 shows the fracture load, mass, and FL/SM of the experimental (left side) and control (right side) samples by site in groups A, B, and C, respectively. The main finding of the present study was that there was no significant difference in sample fracture loads between the frozen-preserved experimental samples
Table 1 Descriptive factors for the skulls in groups A, B, and C. Factor
Group A
Group B
Group C
Number of male skulls Number of female skulls Age range (years) Mean ± SD age (years) Frozen interval of left-side samples Number of all left-side samples Left frontal Left parietal Left medial temporal Left lateral temporal Number of all right-side samples Right frontal Right parietal Right medial temporal Right lateral temporal
22 13 19–95 60.7 ± 20.5 1 day 140 35 35 35 35 140 35 35 35 35
22 13 28–89 66.5 ± 17.0 1 month 140 35 35 35 35 140 35 35 35 35
22 13 24–89 63.5 ± 17.0 3 months 140 35 35 35 35 140 35 35 35 35
Fig. 2. Three-point-bending apparatus.
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S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
and the nonfrozen control samples—regardless of the cryopreservation interval. As shown in Table 2, however, there was a nonsignificant but slight difference in the mass between these samples. As the possibility could not be excluded that the mechanical properties of cranial samples depend on their mass, we devised a way to eliminate any possible bias: The sample’s fracture load was divided by the sample’s mass to determine the FL/SM. The FL/SM of the frozenpreserved sample was then compared with that of its control in each case. The results indicated that there were no significant differences. These findings therefore suggest that the freezing and thawing process has little effect on the mechanical properties of human skull. The findings of this study are in general agreement with results reported in previous studies using bones other than the human skull. Goh et al. reported that freezing at 20 °C for 3 or 21 days had no effect on the mechanical properties of feline long bones [14]. Linde and Sørensen reported that neither storage of trabecular bones by freezing for 100 days nor several thawing, testing, and
refreezing sequences changed the stiffness of the bone [15]. Matter et al. reported that cryopreservation temperatures ( 20 °C or 80 °C) did not have any influence on the mechanical properties of cancellous bone. They also noted that storage duration of up to 2 years had no effect on the mechanical strength of cancellous bone [16]. Panjabi et al. reported that freezing and storage for periods of up to 232 days did not significantly alter the physical properties of cadaver spine specimens [21]. van Haaren et al. reported that there was no effect on the mechanical properties of goat femors or humeri after 20 °C storage for 1 year [17]. In contrast, Sonstegard and Matthews reported a 10% decrease in the stiffness of trabecular bones after frozen storage. They noted that the reason might be trabecular damage caused by expansion of interstitial fluids during freezing [18]. Conversely, Pelker et al. reported a slight increase in bone stiffness after freezing [13]. Similarly, a slight increase in load and the FL/SM of skull samples after frozen storage and thawing were observed in this study. Taken together, changes in the mechanical properties of bone samples after freezing and thawing process have been the subject
Table 2 Differences in fracture load, mass, and FL/SM between the left-side (experimental) and right-side (control) samples by site in 1-day cryopreservation group (A), 1-month cryopreservation group (B), and 3-month cryopreservation group (C).
Fracture load (N)
Site
Group
Left-side samples
Right-side samples
P valuea
All
A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C
505.9 ± 212.7 564.2 ± 241.1 543.1 ± 240.6 702.7 ± 205.8 725.3 ± 217.9 770.7 ± 187.0 439.5 ± 168.2 506.8 ± 196.7 416.0 ± 150.9 450.1 ± 176.4 512.7 ± 238.8 480.4 ± 219.9 431.4 ± 172.8 512.2 ± 242.4 505.4 ± 234.7 5.1 ± 1.2 5.5 ± 1.4 5.4 ± 1.4 5.9 ± 0.9 6.1 ± 1.0 6.4 ± 1.0 4.3 ± 0.9 4.7 ± 1.2 4.3 ± 1.0 4.7 ± 1.0 5.0 ± 1.2 4.8 ± 1.1 5.7 ± 1.1 6.3 ± 1.5 6.0 ± 1.3 98.2 ± 33.1 100.7 ± 32.5 99.6 ± 32.8 123.1 ± 29.7 117.1 ± 26.6 120.5 ± 25.9 100.4 ± 28.0 107.2 ± 27.5 97.2 ± 29.1 94.4 ± 30.5 99.2 ± 34.3 98.4 ± 33.6 75.0 ± 26.1 79.4 ± 29.8 82.3 ± 31.4
489.0 ± 218.7 546.5 ± 240.9 522.9 ± 234.6 699.0 ± 195.5 723.2 ± 212.3 747.8 ± 173.1 420.2 ± 171.8 491.2 ± 198.0 409.4 ± 169.9 425.6 ± 193.2 507.8 ± 250.1 465.2 ± 212.9 411.4 ± 172.7 477.6 ± 201.5 489.3 ± 211.2 5.0 ± 1.1 5.5 ± 1.4 5.4 ± 1.4 5.7 ± 1.0 6.2 ± 1.0 6.3 ± 0.8 4.3 ± 0.9 4.7 ± 1.2 4.4 ± 1.1 4.8 ± 1.0 5.2 ± 1.3 4.9 ± 1.2 5.4 ± 1.1 6.1 ± 1.5 5.8 ± 1.4 95.7 ± 33.6 96.9 ± 32.8 95.4 ± 32.4 122.3 ± 26.2 115.5 ± 28.3 118.9 ± 23.1 96.1 ± 27.3 102.2 ± 22.8 92.4 ± 27.8 89.6 ± 35.8 95.8 ± 38.5 93.8 ± 38.5 74.9 ± 26.4 78.4 ± 25.5 82.4 ± 24.7
0.452 0.606 0.661 0.867 0.755 0.463 0.496 0.690 0.780 0.561 0.856 0.934 0.445 0.496 0.720 0.592 0.872 0.989 0.953 0.751 0.668 0.860 0.760 0.742 0.925 0.561 0.638 0.326 0.625 0.621 0.542 0.408 0.461 0.805 0.729 0.698 0.366 0.408 0.428 0.638 0.537 0.605 0.690 0.819 0.902
Frontal
Parietal
Medial temporal
Lateral temporal
Mass (g)
All
Frontal
Parietal
Medial temporal
Lateral temporal
FL/SM (N/g)
All
Frontal
Parietal
Medial temporal
Lateral temporal
Results are the mean ± SD. FL/SM: fracture load-to-sample mass ratio. a Mann-Whitney U-test.
S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
of some controversy. Linde and Sørensen suggested that before bone storage early postmortem changes lead to altered bone stiffness, especially during the first 24 h [15]. These changes should be considered when interpreting in vitro results. In conclusion, there were no significant differences in sample fracture loads between frozen-preserved (experimental) samples and unfrozen (control) samples regardless of the cryopreservation interval, even when the possibility of sample mass bias was taken into consideration. These results suggest that the freezing and thawing process has little effect on the mechanical properties of human skull. Thus, frozen storage of skulls for up to 3 month is a good preservation method. The results of this study also argue for further research to evaluate the mechanical properties of human skull frozen for longer durations and stored at various temperatures. Ethical standards This study complies with the current laws of the country in which it was performed. Acknowledgments The Designing Everyday Life Function and Social System Team, Digital Human Research Center, National Institute of Advanced Industrial Science and Technology (AIST) supported this work. References [1] Servadei F, Ciucci G, Pagano F, Rebucci GG, Ariano M, Piazza G, et al. Skull fracture as a risk factor of intracranial complications in minor head injuries: a prospective CT study in a series of 98 adult patients. J Neurol Neurosurg Psychiatry 1988;51:526–8. [2] Evans FG, Lissner HR. Tensile and compressive strength of human parietal bone. J Appl Physiol 1957;10:493–7. [3] McElhaney JH, Fogle JL, Melvin JW, Haynes RR, Roberts VL, Alem NM. Mechanical properties of cranial bone. J Biomech 1970;3:495–511.
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[4] Wood JL. Dynamic response of human cranial bone. J Biomech 1971;4: 1–12. [5] Hubbard RP. Flexure of layered cranial bone. J. Biomech 1971;4:251–63. [6] McPherson GK, Kriewall TJ. The elastic modulus of fetal cranial bone: a first step towards an understanding of the biomechanics of fetal head molding. J Biomech 1980;13:9–16. [7] Margulies SS, Thibault KL. Infant skull and suture properties: measurements and implications for mechanisms of pediatric brain injury. J Biomech Eng 2000;122:364–71. [8] Delille C, Baivier S, Masson C, Drazetic P. Identification des lois de comportement du crâne à partir d’essais de flexion. Mécanique & Industries 2003;4:119–23. [9] Delillea R, Lesueura D, Potierb P, Drazetica P, Markiewicza E. Experimental study of the bone behaviour of the human skull bone for the development of a physical head model. Int J Crashworthiness 2007;12:101–8. [10] Coats B, Margulies SS. Material properties of human infant skull and suture at high rates. J Neurotrauma 2006;23:1222–32. [11] Raul JS, Baumgartner D, Willinger R, Ludes B. Finite element modelling of human head injuries caused by a fall. Int J Legal Med 2006;120: 212–8. [12] Topp T, Müller T, Huss S, Kann PH, Weihe E, Ruchholtz S, et al. Embalmed and fresh frozen human bones in orthopedic cadaveric studies: which bone is authentic and feasible? Acta Orthop 2012;83:543–7. [13] Pelker RR, Friedlaender GE, Markham TC. Biomechanical properties of bone allografts. Clin Orthop Relat Res 1983;174:54–7. [14] Goh JC, Ang EJ, Bose K. Effect of preservation medium on the mechanical properties of cat bones. Acta Orthop Scand 1989;60:465–7. [15] Linde F, Sørensen HC. The effect of different storage methods on the mechanical properties of trabecular bone. J Biomech 1993;26:1249–52. [16] Matter HP, Garrel TV, Bilderbeek U, Mittelmeier W. Biomechanical examinations of cancellous bone concerning the influence of duration and temperature of cryopreservation. J Biomed Mater Res 2001;55:40–4. [17] van Haaren EH, van der Zwaard BC, van der Veen AJ, Heyligers IC, Wuisman PI, Smit TH. Effect of long-term preservation on the mechanical properties of cortical bone in goats. Acta Orthop 2008;79:708–16. [18] Sonstegard DA, Matthews B. Mechanical property dependence on storage technique and locale of knee joint trabecular bone. Trans orthop Res Soc 1977;2:283. [19] Nishimoto T, Murakami S, Abe T, Ono K. Mechanical properties of human cranium and effect of cranial fractures on extradural hematoma. Trans Jpn Soc Mech Eng 1995;61:2386–92 (Article in Japanese). [20] Kikugawa H, Asaka T. Effect of long-term formalin preservation on bending properties and fracture toughness of bovine compact bone. Mater Trans 2004; 45:3060–4. [21] Panjabi MM, Krag M, Summers D, Videman T. Biomechanical time-tolerance of fresh cadaveric human spine specimens. J Orthop Res 1985;3:292–300.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Effects of the freezing and thawing process on biomechanical properties of the human skull Suguru Torimitsu a,⇑, Yoshifumi Nishida b, Tachio Takano b, Yoshinori Koizumi b, Mutsumi Hayakawa a, Daisuke Yajima a, Go Inokuchi a, Yohsuke Makino a, Ayumi Motomura a, Fumiko Chiba a, Hirotaro Iwase a a
Department of Legal Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Designing Everyday Life Function and Social System Team, Digital Human Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan b
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 25 November 2013 Accepted 27 November 2013 Available online 7 December 2013 Keywords: Cranial bones Frozen preservation Fracture load Stiffness
a b s t r a c t The aim of this study was to determine if biomechanical investigations of skull samples are reliable after skulls have been subjected to a freezing and thawing process. The skulls were obtained from 105 Japanese cadavers (66 males, 39 females) of known age that were autopsied in our department between October 2012 and June 2013. We obtained bone specimens from eight sites (four bilaterally symmetrical pairs) of each skull and measured the mass of each specimen. They were then classified into three groups (A, B, C) based on the duration of freezing of the experimental samples. The left-side samples were subjected to frozen storage (experimental group). The corresponding right-side samples were their controls. Bending tests were performed on the controls immediately after they were obtained. The experimental samples were preserved by refrigeration at 20 °C for 1 day (group A), 1 month (group B), or 3 months (group C). Following refrigeration, these samples were placed at 37 °C to thaw for 1 h and then were subjected to bending tests using a three-point-bending apparatus attached to a Handy force gauge. The device recorded the fracture load automatically when the specimen fractured. Statistical analyses revealed that there were no significant differences in sample fracture loads between the frozen preserved/thawed samples and the unfrozen controls for each of the cryopreservation intervals. We eliminated any possible sample mass bias by using controls from the same skull in each case. The results suggest that the freezing/thawing process has little effect on the mechanical properties of human skulls. Thus, frozen storage for up to 3 months is a good method for preserving human skulls. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction For forensic analysis of cases involving a fractured skull, it is important to estimate the applied external force. This is because skull fracture is an independent risk factor for intracranial lifethreatening complications such as epidural hematoma, subdural hematoma, and cerebral hemorrhagic contusion [1]. It is also vital to have a sound understanding of the biomechanical properties of the skull so that accurate estimates can be provided. Previous engineering studies have assessed the mechanical properties of human cranial bones using a variety of methods, including compression, tension, and bending tests [2–10]. During the last four decades, biomechanical studies have been conducted in forensic research as well [11]. The importance of such research requires that the skull be preserved in the best possible manner to avoid adding complications to the already difficult determinations.
⇑ Corresponding author. Tel.: +81 43 226 2078; fax: +81 43 226 2079. E-mail address: [email protected] (S. Torimitsu). 1344-6223/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2013.11.005
Some researchers have reported that for human long bones the freezing and thawing process does not significantly alter mechanical properties [12–18]. Hence, fresh human cadaver bones are usually preserved by refrigeration at 20 °C [12]. However, the effects of the freezing and thawing process on mechanical properties of human skull have not been previously studied. The aim of this study was to determine if biomechanical investigations are reliable after freezing and thawing skull samples.
2. Materials and methods 2.1. Subjects The skulls used in this research were obtained from 105 Japanese cadavers of known age and sex that were autopsied at the Department of Legal Medicine, Chiba University, between October 2012 and June 2013. The estimated postmortem interval of each body was within 7 days. Cases were excluded if the history highlighted conditions or events that could have affected the skull.
S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
103
For example, skulls with a fracture, burns, obvious injury, or acquired or congenital abnormality were excluded from this study. The ethics committee of Chiba University approved the study. The skull samples were classified into three groups (A, B, C) as presented in Table 1. 2.2. Sampling During autopsy, bone specimens were obtained from eight cranial sites (four bilaterally symmetrical pairs) from each cadaver skull (left frontal, right frontal, left medial parietal, right medial parietal, left superolateral parietal, right superolateral parietal, left inferolateral parietal, and right inferolateral parietal bones). Fig 1 shows the orientations of samples extracted from each of the cadaver skulls. The length of each specimen was fixed at 50 mm and the width at 10 mm. Left and right frontal specimens were tangential to the left and right side of the coronal suture and along the anterior extended line of the sagittal suture, respectively. Left and right medial parietal specimens were tangential to the left and right side of the coronal suture and along the sagittal suture, respectively. Left and right superolateral and inferolateral parietal specimens were obtained from the lateral portion of the parietal bone immediately superior and inferior to the left and right superior temporal line, respectively. Samples were obtained using an oscillating saw and washed with saline. The mass (grams) of each sample was measured using an electronic scale that measured from 0.1 g to 600 g. A total of 840 samples (420 left-side and 420 right-side samples) were collected. Each of the three groups (A, B, C) comprised 280 samples (140 left-side and 140 right-side samples). The left-side (experimental) samples were subjected to frozen storage. The corresponding rightside samples were their controls in each case. The biomechanical test was performed on the control samples immediately after they were obtained. In contrast, the experimental samples were preserved in a biomedical freezer (MDF-U443; Sanyo, Osaka, Japan) with automatic defrosting function at 20 °C for 1 day (group A), 1 month (group B), or 3 months (group C). Following refrigeration, samples were placed at 37 °C to thaw for 1 h and then underwent biomechanical testing. 2.3. Biomechanical tests Bending tests, which have been used to determine the mechanical properties of various bones including human skull in previous studies [5,19,20], were performed on the skull samples using a three-point-bending apparatus (JSV-H1000; JISC, Nara, Japan) attached to a Handy force gauge (HF-100; JISC) as shown in Fig. 2.
Fig. 1. Orientations of samples extracted from the cadavers skulls.
Two lower supports were set 40 mm apart, and the specimen being tested was placed on the two supports. Above this setup was a 1000-N load cell that applied pressure at the center of the specimen from the outer surface at a constant speed of 100 lm/s. When the specimen fractured, the device automatically recorded the fracture load (N). 2.4. Statistical analysis The fracture load-to-sample mass ratio (FL/SM), in Newtons per gram, was determined by dividing the fracture load by the sample mass. Mann–Whitney U-tests were used to compare the fracture load, mass, and FL/SM between the experimental and control samples by site in each group. Differences of P < 0.05 were considered statistically significant. All statistical analyses were performed on a personal computer using Statistical Package for the Social Sciences (SPSS) version 21.0 software (IBM, Armonk, NY, USA). 3. Results and discussion Table 2 shows the fracture load, mass, and FL/SM of the experimental (left side) and control (right side) samples by site in groups A, B, and C, respectively. The main finding of the present study was that there was no significant difference in sample fracture loads between the frozen-preserved experimental samples
Table 1 Descriptive factors for the skulls in groups A, B, and C. Factor
Group A
Group B
Group C
Number of male skulls Number of female skulls Age range (years) Mean ± SD age (years) Frozen interval of left-side samples Number of all left-side samples Left frontal Left parietal Left medial temporal Left lateral temporal Number of all right-side samples Right frontal Right parietal Right medial temporal Right lateral temporal
22 13 19–95 60.7 ± 20.5 1 day 140 35 35 35 35 140 35 35 35 35
22 13 28–89 66.5 ± 17.0 1 month 140 35 35 35 35 140 35 35 35 35
22 13 24–89 63.5 ± 17.0 3 months 140 35 35 35 35 140 35 35 35 35
Fig. 2. Three-point-bending apparatus.
104
S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
and the nonfrozen control samples—regardless of the cryopreservation interval. As shown in Table 2, however, there was a nonsignificant but slight difference in the mass between these samples. As the possibility could not be excluded that the mechanical properties of cranial samples depend on their mass, we devised a way to eliminate any possible bias: The sample’s fracture load was divided by the sample’s mass to determine the FL/SM. The FL/SM of the frozenpreserved sample was then compared with that of its control in each case. The results indicated that there were no significant differences. These findings therefore suggest that the freezing and thawing process has little effect on the mechanical properties of human skull. The findings of this study are in general agreement with results reported in previous studies using bones other than the human skull. Goh et al. reported that freezing at 20 °C for 3 or 21 days had no effect on the mechanical properties of feline long bones [14]. Linde and Sørensen reported that neither storage of trabecular bones by freezing for 100 days nor several thawing, testing, and
refreezing sequences changed the stiffness of the bone [15]. Matter et al. reported that cryopreservation temperatures ( 20 °C or 80 °C) did not have any influence on the mechanical properties of cancellous bone. They also noted that storage duration of up to 2 years had no effect on the mechanical strength of cancellous bone [16]. Panjabi et al. reported that freezing and storage for periods of up to 232 days did not significantly alter the physical properties of cadaver spine specimens [21]. van Haaren et al. reported that there was no effect on the mechanical properties of goat femors or humeri after 20 °C storage for 1 year [17]. In contrast, Sonstegard and Matthews reported a 10% decrease in the stiffness of trabecular bones after frozen storage. They noted that the reason might be trabecular damage caused by expansion of interstitial fluids during freezing [18]. Conversely, Pelker et al. reported a slight increase in bone stiffness after freezing [13]. Similarly, a slight increase in load and the FL/SM of skull samples after frozen storage and thawing were observed in this study. Taken together, changes in the mechanical properties of bone samples after freezing and thawing process have been the subject
Table 2 Differences in fracture load, mass, and FL/SM between the left-side (experimental) and right-side (control) samples by site in 1-day cryopreservation group (A), 1-month cryopreservation group (B), and 3-month cryopreservation group (C).
Fracture load (N)
Site
Group
Left-side samples
Right-side samples
P valuea
All
A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C
505.9 ± 212.7 564.2 ± 241.1 543.1 ± 240.6 702.7 ± 205.8 725.3 ± 217.9 770.7 ± 187.0 439.5 ± 168.2 506.8 ± 196.7 416.0 ± 150.9 450.1 ± 176.4 512.7 ± 238.8 480.4 ± 219.9 431.4 ± 172.8 512.2 ± 242.4 505.4 ± 234.7 5.1 ± 1.2 5.5 ± 1.4 5.4 ± 1.4 5.9 ± 0.9 6.1 ± 1.0 6.4 ± 1.0 4.3 ± 0.9 4.7 ± 1.2 4.3 ± 1.0 4.7 ± 1.0 5.0 ± 1.2 4.8 ± 1.1 5.7 ± 1.1 6.3 ± 1.5 6.0 ± 1.3 98.2 ± 33.1 100.7 ± 32.5 99.6 ± 32.8 123.1 ± 29.7 117.1 ± 26.6 120.5 ± 25.9 100.4 ± 28.0 107.2 ± 27.5 97.2 ± 29.1 94.4 ± 30.5 99.2 ± 34.3 98.4 ± 33.6 75.0 ± 26.1 79.4 ± 29.8 82.3 ± 31.4
489.0 ± 218.7 546.5 ± 240.9 522.9 ± 234.6 699.0 ± 195.5 723.2 ± 212.3 747.8 ± 173.1 420.2 ± 171.8 491.2 ± 198.0 409.4 ± 169.9 425.6 ± 193.2 507.8 ± 250.1 465.2 ± 212.9 411.4 ± 172.7 477.6 ± 201.5 489.3 ± 211.2 5.0 ± 1.1 5.5 ± 1.4 5.4 ± 1.4 5.7 ± 1.0 6.2 ± 1.0 6.3 ± 0.8 4.3 ± 0.9 4.7 ± 1.2 4.4 ± 1.1 4.8 ± 1.0 5.2 ± 1.3 4.9 ± 1.2 5.4 ± 1.1 6.1 ± 1.5 5.8 ± 1.4 95.7 ± 33.6 96.9 ± 32.8 95.4 ± 32.4 122.3 ± 26.2 115.5 ± 28.3 118.9 ± 23.1 96.1 ± 27.3 102.2 ± 22.8 92.4 ± 27.8 89.6 ± 35.8 95.8 ± 38.5 93.8 ± 38.5 74.9 ± 26.4 78.4 ± 25.5 82.4 ± 24.7
0.452 0.606 0.661 0.867 0.755 0.463 0.496 0.690 0.780 0.561 0.856 0.934 0.445 0.496 0.720 0.592 0.872 0.989 0.953 0.751 0.668 0.860 0.760 0.742 0.925 0.561 0.638 0.326 0.625 0.621 0.542 0.408 0.461 0.805 0.729 0.698 0.366 0.408 0.428 0.638 0.537 0.605 0.690 0.819 0.902
Frontal
Parietal
Medial temporal
Lateral temporal
Mass (g)
All
Frontal
Parietal
Medial temporal
Lateral temporal
FL/SM (N/g)
All
Frontal
Parietal
Medial temporal
Lateral temporal
Results are the mean ± SD. FL/SM: fracture load-to-sample mass ratio. a Mann-Whitney U-test.
S. Torimitsu et al. / Legal Medicine 16 (2014) 102–105
of some controversy. Linde and Sørensen suggested that before bone storage early postmortem changes lead to altered bone stiffness, especially during the first 24 h [15]. These changes should be considered when interpreting in vitro results. In conclusion, there were no significant differences in sample fracture loads between frozen-preserved (experimental) samples and unfrozen (control) samples regardless of the cryopreservation interval, even when the possibility of sample mass bias was taken into consideration. These results suggest that the freezing and thawing process has little effect on the mechanical properties of human skull. Thus, frozen storage of skulls for up to 3 month is a good preservation method. The results of this study also argue for further research to evaluate the mechanical properties of human skull frozen for longer durations and stored at various temperatures. Ethical standards This study complies with the current laws of the country in which it was performed. Acknowledgments The Designing Everyday Life Function and Social System Team, Digital Human Research Center, National Institute of Advanced Industrial Science and Technology (AIST) supported this work. References [1] Servadei F, Ciucci G, Pagano F, Rebucci GG, Ariano M, Piazza G, et al. Skull fracture as a risk factor of intracranial complications in minor head injuries: a prospective CT study in a series of 98 adult patients. J Neurol Neurosurg Psychiatry 1988;51:526–8. [2] Evans FG, Lissner HR. Tensile and compressive strength of human parietal bone. J Appl Physiol 1957;10:493–7. [3] McElhaney JH, Fogle JL, Melvin JW, Haynes RR, Roberts VL, Alem NM. Mechanical properties of cranial bone. J Biomech 1970;3:495–511.
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