Application for human bone identification

These data support previously reported observations that DOs are much more common in humans than nonprimate mammals. How then can this observed difference be applied to the histological identification of bone from archaeological sites that cannot morphologically be distinguished as human or nonhuman? The simplest method is to identify within our data the cutoff point above which the sample must be primate. Prevalence rather than DO count should be used, as it controls for potential species or sample differences in fragment pattern, cortical area, and allometry. Based on our findings, the cutoff is a 1% DO prevalence, above which the sample is almost certainly primate. A 1% DO prevalence could otherwise be stated as a probability that out of 100 secondary osteons sampled, one will be a drifting osteon.

In our sample, only one nonprimate, Lepus californicus, would incorrectly be identified as human using a 1% DO prevalence rate as a cutoff. There are two issues with this sample that may explain its outlier status. First, lagomorphs are notoriously difficult to classify histologically, particularly using standard histomorphological characteristics. For example, they are one of the only nonhuman mammalian species to not develop primary fibrolamellar plexiform bone [23]. Second, the sample is of a zygomatic arch rather than a long bone fragment as is the majority of the sample. Our results suggest there may be a difference in DO prevalence between long bone and cranial elements, and further exploration with a larger sample size is needed to confirm these findings. The only other nonprimate sample with a DO prevalence approaching 1% is from the extinct Lower Eocene carnivore Oxyaena ultimo, an unlikely contributor to archaeological assemblages.

Quantifying the prevalence of drifting osteons can therefore be a useful tool for discriminating human bone fragments. We recommend fragments submitted for this analysis should be from a rib or long bone with a minimum 10% of the cross-sectional area present and the sample is highly unlikely to be from a nonhuman primate given the archaeological context. If the sample is poorly preserved (OHI of <5, then a greater cross-sectional area will be required so that the equivalent of 10% of the total cross-sectional area of the slide is readable. We recommend the following DO criteria are met for a positive identification of human bone: 1) a DO count ≥ 2 and 2) a DO prevalence of ≥ 1% (Fig 5). If these conditions hold, a bone fragment is almost certainly human. Following this method would avoid identifying any nonprimates as human; however, it is also conservative. Only 31 of 124 (25%) humans in our sample would be positively identified as human, but nonhuman fragments would not be incorrectly classified as human.

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Fig 5. Decision tree illustrating how to use the recommended criteria to identify human bone.

https://doi.org/10.1371/journal.pone.0298029.g005

The power of this method is amplified when combined with long-established histomorphological methods for human/nonhuman differentiation, particularly the identification of fibrolamellar plexiform bone and osteon banding in nonhuman bone. The value of documenting the prevalence of drifting osteons is to classify bone fragments as human, but these other well-documented characteristics classify bone fragments as nonhuman. Fibrolamellar plexiform bone is the main primary bone type in large fast-growing mammals. It develops as a fibrous bone scaffolding with later-forming lamellar bone enclosing a web-like vascular plexus that radiates in all directions, often described as having a “brick-like” appearance [24, 25]. Examples of fibrolamellar plexiform bone have been reported in human infants, but are no longer present after the first year of age [26, 27]. This is in stark contrast to most nonprimate mammal bone where large swaths of plexiform bone are present into skeletal maturity and are easily distinguishable from human primary lamellar bone. Osteon banding appears in cross-section as five primary or secondary osteons in a row. While Mulhern and Ubelaker [28] mention observing osteon banding in humans, they point out that it is still useful for distinguishing human and nonhuman bone in applied contexts since human banding is isolated, whereas nonhuman banding generally occurs within plexiform bone or is stacked in several parallel rows. Limited osteon banding has been found in studies of human bone [29, 30]; however, multiple bands of osteons combined with a general linear primary bone structure or coupled with the presence of fibrolamellar plexiform bone remains indicative of nonhuman bone.

The presence of fibrolamellar plexiform bone is therefore the most useful determining factor for identifying younger nonhuman animals that will have transverse cross-sections showing this primary bone structure and very few secondary osteons. Osteon banding is also more common in younger nonhuman individuals, where significant remodeling has not yet obliterated the banded pattern. The limitations of these methods are therefore in identifying nonhumans of advanced age with significant remodeling and positively identifying human bone. Using these methods in conjunction, therefore, will provide the best and most comprehensive results for differentiating human from nonprimate bone.

Other histomorphometric methods such as osteon size and circularity have been evaluated in some limited mammal species with the goal of differentiating human from nonhuman bone in an applied context [18–21]. These studies indicate that human osteons are more variable in size than nonhuman osteons, and there are conflicting reports as to whether human or nonhuman osteons are more circular [31]. Further research on histomorphometric species differentiation, including morphometric analysis of DOs that are often excluded from such studies [e.g. 31], will refine its utility and has the potential to accurately differentiate between nonhuman species.

Effect of age on DO prevalence

DOs have been posited as a distinguishing feature between human adult and juvenile bone [7, 8]. Research indicates an inverse relationship between age and the presence of DOs, with younger individuals having a higher prevalence of DOs [32–34], although DOs are still documented in adult human bone [5, 6, 35, 36]. Streeter [37] published a method of aging human juvenile ribs characterizing the presence of linear DOs as indicative of bone from adolescents. Therefore, age is a key variable to consider in any study of DO prevalence.

Our findings show that membership in the human taxonomic group is a stronger predictor of DO count than age category (Fig 6). Further, contra to previous studies [e.g. 32, 33, 37], we did not find that juvenile humans or nonhumans had a significantly higher count or prevalence of DOs than adult groups. However, our “adult” category was broad, obscuring nuance in different age categories. Recent studies have found DOs highest in the 20- to 30-year age group, classified as “adult” in our study, but that DO osteon population density (OPD) decreased after age 30 [34, 36]. The increase in DO OPD in juvenile and young adult humans may therefore be attributable to growth and development of peak bone mass, which, once achieved, tends to decline steadily into old age. However, DOs are still identified in humans into their nineties [36], so this method may still positively identify humans in any age category.

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Fig 6. Violin plot showing the distribution of DO count for human and nonprimate groups in both age categories.

https://doi.org/10.1371/journal.pone.0298029.g006

Etiology of drifting osteons

What are the potential explanations for the higher occurrence of drifting osteons in human bone? Maggiano et al. [38] demonstrate that remodeling preferentially follows existing vascular canals. The three-dimensional vascular network present in fibrolamellar plexiform bone therefore provides multiple avenues for microcrack repair or other targeted remodeling events. Human bone, on the other hand, has more limited primary vascular pathways, which might necessitate a bone modeling unit (BMU) to “drift” transversely through the cortical space to access areas for targeted remodeling. Andronowski and Cole [39] refer to this as the transverse drift of an osteon “steering” towards a microcrack to repair. Mechanisms for such lateral motion have been proposed previously [40]. Alternatively, the high diversity of secondary osteon morphotypes in primate bone, including DOs, might reflect greater vascular plasticity, allowing for a broader potential variation in vascular connectivity within primate bone relative to nonprimate mammals [41].

Further research is needed to clarify the etiology and function of the drifting osteon morphotype, which may further elucidate the observed taxonomic and age-related differences in DO prevalence. Biomechanical proponents suggest DOs are a result of varying strain placed on the cortex of bone [42]. Strain on the microscopic level is more varied than on the gross level, which may help explain why strain forces cause DOs to form in the same cortex and at the same time as longitudinal secondary osteons [43]. Proponents of metabolic causes postulate that DOs are a consequence of the body utilizing mineral stores within bone at times of stress [44]. However, Epker and Frost [2] discovered no difference in DO frequency between males and females in any age category, which supports a non-metabolic cause for DOs since females in advanced decades typically require more calcium stores with progressing age [45].

Future research directions

Our preliminary findings suggest that the DO prevalence method is unlikely to be a robust metric to differentiate human from nonhuman primate bone (Fig 3), so the method should not be used in contexts where there is potential for nonhuman primate bone as mentioned in Criterion 2 (Fig 5). Follow up research with a larger nonhuman primate sample that controls for age may provide a more nuanced perspective.

Every attempt was made to control for age in our analysis; however, the lack of consistent aging data was a limiting factor. Age data was not available for 103 of 169, or 61% of nonhuman samples. DO prevalence and age could only be evaluated using the broadest age categories of adult versus juvenile. An increased prevalence of DOs has never been documented in nonprimate mammalian juveniles, although this may be due to a lack of research. Further we did not have sufficient data to calculate DO prevalence for the juvenile human group.

As was the case with fibrolamellar plexiform bone and osteon banding [27, 30, 39], it is possible that exceptions will be found within age categories, skeletal elements, or biomechanical outliers in nonhuman bone that may have a higher prevalence of drifting osteons than reported in this study. Additional research is warranted looking at DOs in different age categories of nonhuman mammalian bone, which has received limited attention to date. Similarly, our study was limited in looking at only a few instances of cranial fragments or other flat bones. As reported earlier, the main nonhuman outlier was from a zygomatic arch, supporting the need for further study of all skeletal elements, not only ribs and long bones. It will also be important to confirm using a larger dataset that the prevalence of drifting osteons is comparable across all long bone and rib categories, controlling for age and species.

In summary, this research presents the first method for the histological identification of human bone in applied contexts. In contexts where DNA or proteins are not preserved, such as cremations or tropical environments, this is currently the only method to positively differentiate human from nonprimate bone fragments that cannot be distinguished morphologically. We welcome a blind validation study testing the proposed method using a novel, known-species dataset. Future research is encouraged exploring the addition of additional flat, cranial, and facial skeletal elements and an increased diversity of species to ensure this method can be applied outside of North American contexts.