Introduction

Traumatic brain injury (TBI) is common in forensic actual cases which accounts for the largest proportion of the causes of traumatic death, and it is often accompanied by skull fracture1,2. Studies have shown that finite element models have been developed and applied in various human injury mechanism analysis3,4. The material parameters of finite element modeling of skull usually derive from cadaveric skull experiments. The three-point bending experiment using skull fragments has shown differences in biomechanical parameters of different parts of the skull and the cranial suture in corpses at different ages, thus indicating that the values of the skull and the cranial suture in different parts should be respectively assigned when the finite element model of the skull was constructed5,6. In addition, the skull bones such as frontal, temporal, parietal, and occipital bones are similar to the “sandwich” structure, consisting of the outer plate, diploe, and inner plate7. The outer and inner plates are osteon-dense, and the diploe is cancellous, which microstructures vary greatly. The osteon-dense bone consists of a compact and regularly arranged osteon, while the cancellous bone consists of trabecular bone, which is loose and porous. At present, most finite element models regard the dense/cancellous bone of skull in all parts as a single structure composed of uniform materials. However, studies have shown that the geometry and spatial arrangement of the osteon and trabecular meshwork have great effects on the mechanical properties of the bone8,9,10, which indicates that the biomechanical properties of different parts/layers of the skull may vary. Previous studies have shown differences in mechanical parameters of materials between the outer and inner plates of animal skulls11. Nonetheless, the above studies have not detected the mechanical parameters for skull diploe. Burket et al. found age-related changes in elastic modulus and hardness in animals, manifesting as a sharp increase of the two during rapid bone growth, tending to stabilize during sexual maturation12. However, whether there are differences in biomechanical properties of inner plate, outer plate, and diploe between the skulls and cranial sutures in humans at different ages have not yet been reported. If the above biomechanical parameters were known, the relationship between the shape of skull fracture and injury risk factors such as the magnitude and direction of force could be analyzed more deeply.

The conventional detection methods for biomechanical parameters of the skull, such as a complete skull impact test and a three-point bending test, can hardly accurately detect the skulls with different layers (inner plate, outer plate, and diploe) and can even damage most of the whole sample. Studies have shown that nanoindentation technique has strong advantages in measuring the mechanical properties of materials within different micro-regions, as it can meet the requirements of in-situ and non-destructive testing and can also be used to test samples with small sizes or different shapes13,14,15,16. The material mechanical parameters of human trabecular meshwork such as humerus and femur have been measured using nano-indentation technology. Other studies have shown that nanoindentation technology has a unique value in measuring the bone parameters in tiny areas such as trabecular bone17,18. Therefore, we believed that this method could be used to detect the mechanical parameters of materials in various micro-regions of the skull.

In this study, we explored the feasibility of using nanoindentation to detect the biomechanical parameters in human skulls at different ages, including different parts (frontal bone and coronal suture), and different layers (inner plate, outer plate, and diploe). The purpose is to provide the corresponding reference basis for the construction of a more precise and comprehensive human skull finite element model, and make the model more accurate in the mechanism analysis of craniocerebral injury. At the same time, our study is also important for a comprehensive understanding of the biomechanical research on skull fracture at the micro-level.

Materials and methods

Sample collection

Skull samples (n = 5) were collected from autopsy cases conducted at the School of Forensic Medicine/Forensic Medicine Identification Center of Guizhou Medical University during 2019 (Table 1). All cranial donors were male, and they were divided into five groups according to age, i.e., infant, young child, school-age child, middle-aged and elderly group, with one case per group. Complete personal information such as gender, age, height, and a clear cause of death were available for each skull donor. Note chosen skull donors did not have a skull related complication. Since the freezing process of human hard tissues has little effect on their mechanical properties, the obtained skull samples were unified frozen and stored at the − 20 °C refrigerator for future examination19,20.

Table 1 Skull sample provider information.
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Sample preparation

As shown in Fig. 1, for each skull sample (n = 5), three left frontal (LF) and three coronal suture (CS) pieces with a size of approximately (1~3) cm × 1 cm were cut using an electric cutter. LF-1, LF-2, and LF-3 labeled on LF represented the outer plate (O), inner plate (I), and diploe (D) experimental specimens for nanoindentation detection, respectively. Similarly, each structure of CS was labeled as CS-1, CS-2, and CS-3. Epoxy resin and toughening curing agent were uniformly mixed with a mass ratio of 100:40 and then poured into a cylindrical soft silica gel mold with an inner diameter of 30 mm. Thawed samples were placed in the prepared gel with the experimental side down at room temperature until completely solidified. Next, low-mesh to high-mesh metallographic sandpapers (mesh numbers: 800, 1200, 1500, 2000, and 4000) were sequentially used to grind and polished on a metallographic sample polishing machine (MPD-1) at a rotating speed of 500 r/min until there were no obvious scratches on the surface of the samples. Until all areas on the experimental surface of the sample could be clearly observed under the microscope with the same magnification (Fig. 2).

Figure 1
figure 1

Material drawing of each part of the skull.

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Figure 2
figure 2

Physical diagram of CS samples at different ages. Samples (a, b, c, d, and e) were 0.047, 3, 8, 51, and 77 years of age, respectively.

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Thickness measurement

Prior to the nanoindentation measurements, thickness of the considered sections was measured under the microscope configured to the nanoindentor. The thickness of the inner plate, the diploe and the outer plate of the sections were obtained as well as the total thickness. The total thickness, the thickness of the inner plate, the diploe and the outer plate were obtained into three values. The mean value and the standard deviation were obtained through calculation.

Nanoindentation experiment

Experiments were performed using an NHT3 (Anton-Paar, PESEUX, Switzerland) nanoindentation apparatus. The tip of the ram was loaded into the sample at 40 mN/min for 10 s at a maximum load of 20 mN and then loaded out at 40 mN/min. As shown in Fig. 3, nine points were selected on the the outer plate and the inner plate surface of the skull (LF-1, LF-2), and the midpoint between the outer plate and the inner plate surface of the skull (LF-3, where is cancellous bone) for nano indentation detection. Of the above nine points, the distance between adjacent points is 100 μm. In the actual detection process, considering the weak compression ability of CS and the limitation of nanoindentation technology, some selected detection indentations were located in CS, while most of them were on both sides of CS. During the test, the computer detection software automatically outputs loading and contact areas to calculate Brinell hardness (HIT in MPa) and modulus of elasticity (E in GPa). The method proposed by Oliver and Pharr et al. was used to obtain the modulus of elasticity (EIT in GPa)21 (assuming v = 0.3 poisson's ratio).

Figure 3
figure 3

Bitmap of nanoindentation points of inner plate, diploe and outer plate of the LF.

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Statistical analysis

Descriptive results were presented primarily as means and standard deviation (SD). All statistical calculations were performed by IBM SPSS statistical software (SPSS, Version 25, IBM, Armonk, New York). P < 0.05 was considered statistically significant. One-way and multi-way ANOVAs were used to test the main effect contributions of age, structure, and location, as well as the interaction between E and HIT.

Ethical considerations

This study was approved by the Ethics Committee of Guizhou Medical University (approval 2021 No.1). Informed consent was obtained from the parents and/or legal guardians for the use of the skull specimen in this study. All experimental procedures and methods were performed in accordance with approved guidelines and standards applicable to the forensic context.

Results

Total thickness of each layers of LF and CS

With age increasing, the total thickness of the LF and CS gradually increased, reaching the maximum in the 51 years of age group and then decreasing slightly (Fig. 4a). The thickness of LF was higher than that of CS (except for 0.047 years of age group).

Figure 4
figure 4

Change trend diagram of thickness of total and different layers of LF and CS at different ages. The (a) is the thickness of the whole skull, and (b) is the thickness of each layer of the skull inner plate, the diploe and the outer plate.

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The results of different layers of LF and CS showed that the thicknesses of the inner plate, diploe and outer plate tended to increase with age, and the thicknesses of LF and CS diploe were higher than those of the inner plate and outer plate (Fig. 4b).

Effects of age, layers, and location on HIT and E

Descriptive results are mainly reported as the mean and the standard deviation (SD) (Tables 2, 3). Overall, results showed that there are statistical differences in HIT (P < 0.001) and E (P < 0.001) among the considered age groups. In Fig. 5a, the left Y-axis represents HIT, and the right Y-axis represents E. HIT and E showed an increasing trend before 51 years of age and a slightly decreasing trend after 51 years of age. HIT between the inner, outer plates and diploe were not correlated (P = 0.835), while E exhibited a significant trend (P = 0.056) and diploe E were higher than that of the outer and inner plates (Fig. 5b). There were significant differences in HIT and E of each site between groups (P < 0.05), and HIT and E of LF were significantly higher compared to those of CS (Fig. 5c).

Table 2 HIT single-factor ANOVA.
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Table 3 E single-factor ANOVA.
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Figure 5
figure 5

One-way analysis of variance of HIT and E based on different ages, layers and locations. The (a) is the HIT and E changing trend of LF and CS at different ages; (b) is the E difference of different structures of inner plate, diploe and outer plate; (c) is the difference comparison of different parts (LF and CS).

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Effect of combination of age, layers and location on HIT and E

Multi-factor ANOVAs determined the main effect contribution of age, layers and location and their interactions on E and HIT. In Table 4, HIT of age (P < 0.001) and location (P < 0.001) had statistically significant, while HIT of the layers had no correlation (P = 0.487). In Table 5, E of age (P < 0.001), layers (P < 0.001) and location (P < 0.001) had statistically significant. The same age and structure, HIT and E statistical analysis of location were shown in Tables 6 and 7, respectively. Same as the result of single-factor ANOVAs, HIT showed an upward trend before 51 years of age and a downward trend after 51 years of age, and difference from the results of single-factor ANOVAs, E decreased from 0.047 to 3 years of age, then increased to 51 years of age, and finally continued to decrease to 77 years of age (Fig. 6a). Diploe E was higher than that of the outer and inner plates (Fig. 6b). And HIT and E of LF were significantly higher than that of CS (Fig. 6c). Figure 7 showed the same layers and location, age and location, statistical description of the age, layers were displayed. HIT and E for both CSD and LFD showed results consistent with a one-way analysis of variance that were maximal in the 51 years of age group (Fig. 7a,c). The same age and location, we found higher E of plate in the 0.047, 8, 51, and 77 years of age groups of LF, and in the 0.047, 8, and 77 years of age groups of CS than in the inner and outer plates, which was consistent with the results of a one-way analysis of variance (Fig. 7d).

Table 4 HIT tests of between-subjects effects.
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Table 5 E tests of between-subjects effects.
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Table 6 HIT comparison among different locations.
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Table 7 E comparison among different locations.
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Figure 6
figure 6

Multi-way analysis of variance of HIT and E based on different ages, layers and locations. The (a) is the HIT and E changing trend of LF and CS at different ages; (b) is the E difference of different layers of inner plate, diploe and outer plate; (c) is the difference comparison of different parts (LF and CS).

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Figure 7
figure 7

Multi-way analysis of variance of HIT and E based on different ages, layers and locations. The (a) and (c) show the statistical description of HIT and E of ages at the same layers and location. The (b) and (d) show the statistical description of HIT and E of location at the same layers and ages. And the group with statistical significance are connected by straight lines.

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Correlation analysis between E and HIT

Pearson correlation coefficient was used to identify the correlation between E and HIT measured at different ages, in different layers and different parts. The results showed that both HIT and E were correlated with age (P < 0.05, P < 0.05) and location (P < 0.05, P < 0.05), while neither HIT nor E was correlated with layers (P > 0.05) (Table 8).

Table 8 The correlation coefficients between E and HIT by Pearson.
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Discussion

Our results showed that HIT and E differed in all age groups, reaching the maximum in the middle-aged group and then slightly decreasing. The reason for this may be that human skeleton, including the skull, continuously grows and changes. From infants to young children, bone growth has a dominant role, after which it tends to enter a state of homeostasis for a period of time in adulthood, while bone quality and bone mass gradually decrease with age22. In this study, the correlation between age and layers was analyzed, and the result was positive, which further verified the correlation between each biomechanical parameter and age. Additionally, the difference in HIT for each layers was not statistically significant. E of diploe was higher than that of the inner plate and the outer plate. Further correlation analysis revealed no correlation between HIT/E and structure. This result might indicate that the osteon of the inner plate and the outer plate had the same or similar hardness as the trabecular meshwork constituting the diploe, and there was no difference in compression resistance.

Previous studies have reported differences in biomechanical properties of different parts of skulls. The main research method used in these studies was the three-point bending experiment of skull slices, and the research content was generally the detection of elastic modulus and bending strength. The results showed biomechanical differences among various parts, including cranial sutures5. In this study, the HIT and E of the left frontal and coronary sutures were studied using nanoindentation technology. Our results revealed a difference between the cranial and cranial sutures, and the HIT and E of the left frontal were higher than those of the coronary suture. Cranial suture continues to develop after birth, while it is not completely closed in infancy. With aging, the cranial tissues on both sides of the suture grow, continuously develop, and then gradually close23. In this study, the sampling location of the cranial suture was set as the skull adjacent to the cranial suture, which was different from the biomechanical parameters of the skull, thus indicatting that the biomechanical properties of the cranial tissue grown in the late period around the cranial suture were lower compared to other cranial tissues. Moazen et al. measured the biomechanical parameters of the mouse skull using nanoindentation experiment. Consistent with the results of this study, the elastic modulus of the mouse cranial suture was also lower than that of the skull24. These results indicated that the assignment of values to the cranial tissues around the cranial sutures should be made differently when the finite element model of the skull was constructed. Our study showed the correlation between HIT and E of the left frontal and coronary sutures with the change of age, indicating that each part of the skull grew to different degrees with aging, which was consistent with the results of the previous study25,26.

The results of this study showed that the thickness of LF/CS changed with ages. The peak was also in the middle-aged group and decreased to the elderly group, which was consistent with the change trend of HIT and E, coinciding with the research conclusion of Torimitsu et al., whose study showed that skull thickness correlated with biomechanical parameters5.

In summary, the present study successfully used the nanoindentation technique to measure the biomechanical parameters of various human skull layers from different parts of the human body at different ages. Our results revealed differences in the micromechanical parameters of the human skulls at different ages compared with the inner, diploe, and outer plates of the cranial sutures. The biomechanical properties of human skulls were related to age, location, and layers. Our research suggests that, when constructing the refined human head/skull finite element model, the properties and parameters of skull materials at different ages, in different parts (skull/cranial suture), and in different layers (outer plate, inner plate, and diploe) should be assigned with different values.

Conclusion

The biomechanical properties of human skull/cranial suture with different ages and different layers are different. The HIT and E of skull are higher than those of cranial suture, and the diploe E is higher than the inner and outer plates. The differences of material mechanical parameters of age, layers and location should be considered in the finite element modeling of skull.