EVALUATING DIFFERENTIAL NUCLEAR DNA YIELD RATES AMONG HUMAN BONE TISSUE TYPES: A SYNCHROTRON MICRO-CT APPROACH

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone is suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this research is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types, with a focus on osteocytes and the 3D quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Methods included: (1) quantifying cortical and cancellous bone volume from each bone-sampling site using Computed Tomography (CT), and (2) visualizing and quantifying osteocyte lacunae using synchrotron radiation micro-Computed Tomographic imaging (SR micro-CT). Regions of interest (ROIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren’s (2014) study. Analyses tested the primary hypothesis that the abundance and density of bone’s cellular spaces vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone ROIs containing a higher lacunar abundance and density. The osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from predominantly cancellous bones. It is hypothesized that soft tissue remnants within the medullary cavities of primarily cancellous skeletal elements are driving the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context. The procurement of small, primarily cancellous bones with associated soft tissues should be preferentially sampled, and no longer dismissed as potential DNA sources in favor of cortical bone tissue

Age-Related Changes to Bone Microarchitecture in a Non-weight Bearing Bone

This study explored the relationship between age, sex and bone microarchitecture. Using high-resolution images, we may better understand the factors affecting different aspects of bone microstructure. Statistical support was provided for the quantitative differences in bone microarchitecture between the sexes. The significant differences noted in Canal Volume (Ca.V), Cortical Porosity (Ca.V/TV), Canal Surface (Ca.S) and Canal Surface to Tissue Volume (Ca.S/TV) offer several interesting points when compared with pre-existing literature. All four parameters listed above that reported significance did so with respect to sex, in that each parameter differed between sexes. However, no significant difference was noted among age within either sex. The biggest problem regarding the efficacy of this study was the small sample size for each age group. For any given age group, the sample size was not larger than 2 for each sex. This made it difficult to determine the effects of age on bone microstructure within one sex quantitatively. This may be due to the large of variation in the parameters we tested for. Support for age-related changes in bone microarchitecture within one sex, and additional support for changes between sex may be provided in the future when using a larger sample size

Recent advancements in the analysis of bone microstructure: new dimensions in forensic anthropology

Bone is a mechanically active, three-dimensionally (3D) complex, and dynamic tissue that changes in structure over the human lifespan. Bone tissue exists and remodels in 3D and changes over time, introducing a fourth dimension. The products of the remodelling process, secondary and fragmentary osteons, have been studied substantially using traditional two-dimensional (2D) techniques. As a result, much has been learned regarding the biological information encrypted in the histomorphology of bone, yielding a wealth of information relating to skeletal structure and function. Three-dimensional imaging modalities, however, hold the potential to provide a much more comprehensive understanding of bone microarchitecture. The visualization and analysis of bone using high-resolution 3D imaging will improve current understandings of bone biology and have numerous applications in both biological anthropology and biomedicine. Through recent technological advancements, we can hone current anthropological applications of the analysis of bone microstructure and accelerate research into the third and fourth dimensional realms. This review will explore the methodological approaches used historically by anthropologists to assess cortical bone microstructure, spanning from histology to current ex vivo imaging modalities, discuss the growing capabilities of in vivo imaging, and conclude with an introduction of novel non-histological modalities for investigating bone quality

Comment on “assessing the extent of bone bioerosion in short timescales – A novel approach for quantifying microstructural loss [Quat. Int., in press https://doi.org/10.1016/j.quaint.2023.01.011]”

We outline issues with a recent publication by Mein and Williams (2023), which is primarily based on the morphology and quantification of osteocyte lacunae, microscopic cavities that house cells (osteocytes) which sustain bone tissue in living vertebrates. Using 26 rats (Rattus rattus) of unreported age and sex, the authors propose a method whereby post-mortem bone degradation on a short timescale can be quantified by analysing subtle shape changes of osteocyte lacunae. However, a lack of consideration is given to the natural variation and other biological factors influencing osteocyte lacunar morphology; the proposed diagenetic composition of lacunae is not determined; and the methods employed, including the animal model used, are unfit for the purpose of the study. Given these issues, we believe the conclusions presented by the authors should be treated cautiously and not extended to forensic investigations of post-mortem interval, particularly in humans

Characterizing the postmortem human bone microbiome from surface-decomposed remains.

Microbial colonization of bone is an important mechanism of postmortem skeletal degradation. However, the types and distributions of bone and tooth colonizing microbes are not well characterized. It is unknown if microbial communities vary in abundance or composition between bone element types, which could help explain differences in human DNA preservation. The goals of the present study were to (1) identify the types of microbes capable of colonizing different human bone types and (2) relate microbial abundances, diversity, and community composition to bone type and human DNA preservation. DNA extracts from 165 bone and tooth samples from three skeletonized individuals were assessed for bacterial loading and microbial community composition and structure. Random forest models were applied to predict operational taxonomic units (OTUs) associated with human DNA concentration. Dominant bacterial bone colonizers were from the phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes. Eukaryotic bone colonizers were from Ascomycota, Apicomplexa, Annelida, Basidiomycota, and Ciliophora. Bacterial loading was not a significant predictor of human DNA concentration in two out of three individuals. Random forest models were minimally successful in identifying microbes related to human DNA concentration, which were complicated by high variability in community structure between individuals and body regions. This work expands on our understanding of the types of microbes capable of colonizing the postmortem human skeleton and potentially contributing to human skeletal DNA degradation

Cortical bone porosity in rabbit models of osteoporosis

Cortical bone porosity is intimately linked with remodeling, is of growing clinical interest, and is increasingly accessible by imaging. Thus, the potential of animal models of osteoporosis (OP) to provide a platform for studying how porosity develops and responds to interventions is tremendous. To date, rabbit models of OP have largely focused on trabecular microarchitecture or bone density; some such as ovariectomy (OVX) have uncertain efficacy and cortical porosity has not been extensively reported. Our primary objective was to characterize tibial cortical porosity in rabbit-based models of OP, including OVX, glucocorticoids (GC), and OVX + GC relative to controls (SHAM). We sought to: (i) test the hypothesis that intracortical remodeling is elevated in these models; (ii) contrast cortical remodeling and porosity in these models with that induced by parathyroid hormone (1–34; PTH); and (iii) contrast trabecular morphology in the proximal tibia across all groups. Evidence that an increase in cortical porosity occurred in all groups was observed, although this was the least robust for GC. Histomorphometric measures supported the hypothesis that remodeling rate was elevated in all groups and also revealed evidence of uncoupling of bone resorption and formation in the GC and OVX + GC groups. For trabecular bone, a pattern of loss was observed for OVX, GC, and OVX + GC groups, whereas the opposite was observed for PTH. Change in trabecular number best explained these patterns. Taken together, the findings indicated rabbit models provide a viable and varied platform for the study of OP and associated changes in cortical remodeling and porosity. Intriguingly, the evidence revealed differing effects on the cortical and trabecular envelopes for the PTH model.</p