Computed tomography differentiation of compact and cancellous bone tissue in short and sesamoid bones

Good and dependable sample selection of human skeletal remains for genetic analysis has been one of the main goals of many studies in recent years.^1–11 Some explore the issue of bone types, their structure and varying amounts of DNA in different anatomical regions using knowledge from embryology, histology, and widely known scientific facts. In contrast, others try to explain the variations in DNA quantity by studying the effects of external factors on DNA preservation.^1,4,12

Bones are classified into seven groups based on their shape, size, and thickness. Bone tissue is further categorised into compact and cancellous bone, which merge without clear boundaries. While the structure of long bones is relatively straightforward, the structure of other bone types, especially short and sesamoid bones, is more complex. These bones consist of thin-to-medium-thick layers of compact bone and varying amounts of cancellous bone.¹³ It was recently found that cancellous bones, such as small bones of hands and feet, contain more significant amounts of DNA than compact long bones.⁴ The petrous bone in the skull is the skeletal element with the most preserved DNA in ancient remains^14–19 and forensic skeletal investigations.^14–16 Selecting the right skeletal element for genetic testing is crucial, as the petrous bone may not always be available. When only short and sesamoid bones are accessible, determining which part of the bone should be used for DNA extraction becomes essential. Regardless of the bone type chosen intra-bone DNA preservation varies significantly, affecting STR typing success rates. Inter-bone DNA yield depends on bone composition, particularly the ratio of cortical to cancellous tissue, which differs across skeletal elements.^{4,6–8,17,18}

To determine the best DNA sampling sites, specific examination techniques can be used, such as Attenuated Total Reflectance (ATR), Fourier Transform Infrared (FTIR) spectroscopy, and micro-computed tomography (micro-CT).^19,20 ATR-FTIR provides detailed information about the chemical composition of bone from the surface down to a few micrometres in depth, while micro-CT offers an extremely detailed view of the microstructure of bone samples. Both techniques are insightful, however they do not allow for a comprehensive study of the bone in its entirety.

In this study, we utilised dual-source computed tomography (DSCT), a relatively new technique that can generate high-quality images of soft tissue or bone marrow by distinguishing different tissue characteristics to ensure precise and effective DNA sampling from dry, skeletonised human remains.^21–24

Our study aimed to integrate DSCT into targeted DNA sampling to determine compact and cancellous bone regions in small skeletal elements for DNA extraction optimisation.

Using DSCT, an already established method of measuring bone density in the general living population, we successfully mapped out the internal structural differences of dry, dead, and skeletonised human remains. Excavation from the same grave enabled comparison of bones from different skeletons, all exposed to identical decomposition and environmental conditions.

This study highlights the interesting and complex internal structure of the six small skeletal elements that we examined. CT imaging provided invaluable insight, identifying distinct bone regions that would have otherwise been overlooked. Our study focused on the smaller bones of the lower extremity. Without CT imaging, these bones may have been mistakenly divided in half, which would have mixed their compact and cancellous parts during further genetic analysis. Our results demonstrate that the internal structure of short and sesamoid bones is individual, and each skeletal element is unique. It does not always follow the belief that bones thrive under pressure, resulting in tougher and denser parts due to remodelling, microtraumas, and repeated mechanical stresses.^30–32 Some bones had one or multiple bone islands (1 patella, 6 calcanei, 4 tali, 7 cuboid bones, 11 navicular bones and 1 medial cuneiform bone), and 3 of the anterior surfaces of patellae had prominent bony outgrowths representing proliferative enthesopathy of the quadriceps muscle.

Our study found differences in bone density among the compact parts of the calcaneus, the navicular bone, and the medial cuneiform bone. Specifically, the bone density was higher in the calcaneal sulcus than in the other compact parts of the same bone (anterior and posterior processes). The calcaneal sulcus is located in the tarsal sinus, a cylindrical cavity between the talus and calcaneus containing blood vessels, nerves, fat, and numerous ligaments crucial for ankle stability during eversion and inversion. In addition, we found that the navicular bone’s densest part is the proximal articular surface, which articulates with the talus’s head. This density may result from the physical load transfer, which results in remodelling and repairing microtraumas. According to our findings, the density of the medial cuneiform bone’s medial surface is higher than its proximal articular surface, which is likely due to the tibialis anterior muscle insertion site being on the medial surface. Our findings confirm significant intra-bone variability in bone density, even within the same type of bone structure.

This research represents a convergence of the fields of radiology and forensics. In the literature we came across, some researchers have analysed bone structure using detailed anatomy to identify individual-specific alterations or changes throughout historical periods using micro-CT and CT.^33–41 Others have used CT to estimate age-at-death, determine sex and stature, and explore potential implications for forensics, either on forensic autopsy cases or living individuals.^42,43 Currently, micro-CT is the most interesting and commonly used method for investigating intra-bone variability in DNA. Micro-CT allows imaging of small-sized samples (ranging from 100 nanometres to 200 millimetres) with excellent resolution, providing insights into morphology investigations. However, despite its advantages, micro-CT has certain limitations. One such disadvantage is that it is complex, demanding, and not widely available for research work. Additionally, the method does not allow for the examination of the bone in its entirety.

To the authors’ knowledge, this study is the first to analyse bone density within dry, skeletonised human remains to identify possible intra-bone variability using DSCT, an already established method of measuring bone density in the general living population. Our results provide insight into the specific internal structure of six skeletal elements as a guide for targeted DNA sampling selection.

We could only use well-preserved bones without cracks or missing areas for our material selection. As a result, some skeletal elements had fewer bones to evaluate, with only 12 talar bones and 15 patellae available. Regardless, the trend of the structure of the bones was sufficiently seen in the number of bones observed. It is possible to divide these six skeletal elements into compact and cancellous bone tissue by slicing them into thicker slices with a saw and simply observing the structure within. However, this approach could result in inconsistent slices, and valuable DNA information could be lost as parts of the bone are sawed away. Therefore, we decided not to pursue this method to avoid losing important information for subsequent further DNA analysis. While analysing CT images, we observed a simple fact: we identified more numerous but smaller, compact regions and larger cancellous regions, resulting in more compact regions per skeletal element.

Our study successfully determined areas of compact and cancellous parts of 6 specific dry, dead bones using DSCT, an already established method of measuring bone density in the general living population. The bone structure is essential for determining the best sampling site for DNA extraction for molecular genetic identification in forensics and paleogenetics. Our future work could assess whether a correlation exists between CT-measured bone density and the amount of preserved DNA of small skeletal elements.