Are Teeth Better? Histological Characterization of Diagenesis in Archaeological Bone–Tooth Pairs and a Discussion of the Consequences for Archaeometric Sample Selection and Analyses

Teeth are often the preferred source material for isotopic and genetic assay involving ancient biomolecules. The assumption is that dental tissue preserves better due to its anatomically protected location, the enamel cap, and lower porosity compared to bone. However, this assumption has not been widely tested. Some similarities in diagenetic processes are to be expected due to similarities in structure and chemical composition of dentine and bone. This has led to the suggestion that bone can be used as an indicator of dental preservation, as a pre‐screening technique in the selection of suitable samples for biomolecular studies. Thus, direct testing of the correlation between bone and tooth preservation and diagenesis is needed. This paper reports the results of the histological characterization of diagenetic alterations within 25 human femur–tooth pairs, from a Medieval to modern (AD 1850) cemetery in Eindhoven, the Netherlands. The results showed that teeth do indeed preserve better overall, but not always, and that this was dependent on the main diagenetic factor(s) at the burial location. Furthermore, good correlations are found between the microstructural preservation of bone and teeth; similar processes of decay were observed within bone and teeth of the same individual. Overall, the study demonstrated that histological analysis of bone is useful for the identification of degradation processes that affect biomolecular preservation in skeletal material. In this way, sample selection and analytical strategies can be optimized. Copyright © 2013 John Wiley & Sons, Ltd.     

ORGANIC RESIDUE ANALYSIS IN ARCHAEOLOGY: THE ARCHAEOLOGICAL BIOMARKER REVOLUTION*

Organic residue analysis utilizes analytical organic chemical techniques to identify the nature and origins of organic remains that cannot be characterized using traditional techniques of archaeological investigation (because they are either amorphous or invisible). The field is founded upon the principle that the biomolecular, or biochemical, components of organic materials associated with human activity survive in a wide variety of locations and deposits at archaeological sites. The archaeological information contained in organic residues is represented by the biomolecular components of the natural products that contribute to the formation of a given residue. By applying appropriate separation (chromatographic) and identification (mass spectrometric) techniques, the preserved, and altered, biomolecular components of such residues can be revealed. Once identified, the Archaeological Biomarker Concept can be applied, wherein the structure and even isotopic composition(s) of a given biomolecule or suite of biomolecules (the ‘chemical fingerprint’) can be related to the compositions of organisms exploited by humans in the past. As the organic residue field emerges from its pre‐paradigmatic phase, and the organic residue revolution gathers pace, the way is open for challenging many long‐held archaeological hypotheses and offering new perspectives on the study of human activity in the past.     

Bone Degradation and Environment: Understanding,Assessing and Conducting Archaeological Experiments Using Modern Animal Bones

Archaeological experiments that use modern bones to replicate past animal bone assemblages have often failed to consider the effects of environment, storage and preparation on modern bones. Often, these experiments make little mention of the conditions to which bones were subject during their storage and preparation for use in experiments. In other instances, these variables are reported but not considered as factors that contribute to the nature of the results obtained. This study considers previously reported data concerning the degradation of frozen bones (−20°C), and bones exposed to hot, dry conditions (40°C), and presents new data for bones exposed to room temperature environments (22°C) and refrigerated environments (2°C), and bones that are frozen (−20°C) and then thawed (22°C). These conditions are all relevant to understanding the nature of bone degradation and the use of bones in modern archaeological experimentation. This article also surveys a range of previously reported experiments that utilise modern bones to create analogies to the past and considers different methodological approaches and their relationship to the condition of bones at the time of their fracture and fragmentation. The longitudinal data presented in this study demonstrate differential rates of bone degradation over time in various environmental conditions. This degradation results in dramatic changes in bone fracture morphology, bone strength and utility for bone tool production. These observations have significant implications for experiments that utilise modern bones, especially when experimental data are used to create analogies to the archaeological past. Copyright © 2012 John Wiley & Sons, Ltd.     

The crystallinity of ancient bone and dentine: new insights by transmission electron microscopy

We studied various archaeological and palaeontological bones and dentines from different burial environments by Fourier transform infrared spectroscopy (FT–IR), X–ray diffraction (XRD) and transmission electron microscopy (TEM), in the framework of a general study of diagenesis. FT–IR and XRD were used to evaluate the global preservation state of the bone and dentine mineral phase by determining a splitting factor (SF) or a crystallinity index (CI), respectively. These data can be combined with studies on the nanometer scale made with TEM. This latter technique,coupled with electron microdiffraction, provides determination of dimensions and shapes of individual bone and dentine apatite nanocrystals as well as of secondary minerals formed during diagenesis. It enables us to distinguish between heat–induced recrystallization processes and crystal growth in solution occurring during diagenesis.     

Experimental effects of bone size and temperature on bone diagenesis

In the ground, bone undergoes chemical and physical changes which affect its preservation. This fact has important implications for dating and other analytical procedures involving bone, as well as faunal analysis where differential preservation of bones of different species may affect conclusions regarding the relative significance of an animal to the economy of a given society. The diagenic processes in bone range from minor changes in the bone protein to complete structural and chemical breakdown.Using fresh cow bone, we conducted laboratory experiments which simulate the effect of temperature and bone size on the rate and nature of bone disintegration in archaeological sites. Temperature influences the rate of chemical change, and bone size and density affect the accessibility of the molecular constituents of bone to extrinsic chemical reactions. These findings clarify the importance of two well-known concepts in bone taphonomy. (1) The rate of chemical breakdown in bone tissues is related to the proximity of a given unit of tissue to the bone surface. This means that, in archaeological bone samples, tissue near the surface may be different chemically from tissue away from the surface and great care is necessary in choosing and preparing bone samples for analytical procedures. (2) In general, small bones are not as well preserved as large bones, therefore small animals are likely to be underrepresented in faunal assemblages.     

Pick the Right Pocket. Sub‐sampling of Bone Sections to Investigate Diagenesis and DNA Preservation

Many archaeological bones display a heterogeneous degradation pattern. Highly degraded bones could contain pockets of well‐preserved bone, harbouring good quality DNA. This dichotomy may explain why the relationships between global bone preservation parameters such as histological integrity, bone mineral crystallinity or collagen yield, and bulk DNA preservation/amplification success rate have been found to be at best, weak to moderate. In this pilot study, we explore whether or not a more localised approach will highlight a stronger relationship between diagenetic parameters and DNA preservation. This study includes a detailed histological characterisation of bone diagenesis in sub‐areas of three bone samples. Regions of the same bone, which displayed differential degrees of preservation or type of diagenesis were sampled for further analysis and both genetic (small scale Illumina MiSeq sequencing) and chemical (Fourier‐transform infrared spectrometric analysis) analyses were performed. The aim was to investigate how bone diagenetic processes relate to DNA preservation at a higher resolution than in previous studies. This is key in order to improve DNA analytical success rates. The expected relationship between bone and DNA preservation (retrieved endogenous DNA) was observed and the results corroborate previous work that DNA preservation is linked to the integrity of bone collagen and mineral. The results further suggest that non‐biological diagenetic alterations such as etching and the presence of mineral infiltrations and inclusions have a negative effect on DNA preservation/extraction. Copyright © 2016 John Wiley & Sons, Ltd.     

骨角质文物保护研究进展

骨角质文物丰富的历史价值使其成为世界各地考古学、人类学、艺术史研究的重要材料,但由于骨角质文物一般年代久远、保存环境各异以及自然矿化等因素造成该类文物的保存状况不容乐观。该文拟从骨角质文物保护研究的角度,对目前骨角质文物的制作材料与工艺、病害研究、修复技术及预防性保护等方面进行梳理,并尝试找出目前存在的问题与保护需求。我国骨角质文物保护需求主要包括出土饱水器物的现场保护是骨角质文物保护中亟待解决的问题;中国最早的古文字资料即有字甲骨的防风化研究是骨角质文物保护中的重点课题;全国各地出土的不同时期动物骨骼的保护是骨角质文物保护工作的主要内容。     

Bone diagenesis in the European Holocene II: taphonomic and environmental considerations

We have applied cluster analysis to mercury intrusion porosimetry data from 219 archaeological bones (121 human and 98 animal) and soil chemistry data from 219 accompanying soil samples (1 per bone sample), to investigate the influence of soil chemistry on bone preservation. The samples chosen for the study were obtained from sites ranging in time from the pre-modern to the Mesolithic and were representative of burial environments across Europe (from the Baltic to the Mediterranean). These results represent the single largest database for archaeological bone preservation in the European Holocene to date and demonstrate the potential for large-scale diagenetic studies to help develop long term preservation strategies for our European heritage. Despite the variety of sites and environments, bones could be categorised into only four main diagenetic types. Furthermore, soil chemistry appears to significantly affect only one type of preservation, the pathway characterised by loss of mineral. In neutral to basic soils, taphonomy and in particular the differences between the treatment of human and animal remains, becomes the dominating factor in determining preservation. Using these results, strategies for heritage management of archaeological sites can be suggested; grouping sites into those requiring immediate excavation and those where in situ preservation is viable.     

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones.     

Biological mineral content in Iberian skeletal cremains for control of diagenetic factors employing multivariate statistics

The aim of this study was to define a strategy for a correct selection of bone samples by employing inductively coupled plasma optical emission spectroscopy (ICP-OES) for reconstructing the biological mineral content in bones through the determination of major elements, trace elements and Rare Earth Elements (REE, lanthanides) in skeletal cremains of ancient Iberians (III–II B.C), discovered in the Necropolis of Corral de Saus (Moixent, Valencia) between 1972 and 1979. The biological mineral content was determined taking into account diagenetic factors. A control method for a better reading of results was applied. To explore large geochemical datasets and to reduce the number of variables, Principal Component Analysis (PCA) was used, thus, providing a deeper insight into the structure of the variance of the dataset. PCA shows that the elemental profiles of bone and soil samples are clearly different. Bone samples obtained from the outer bone layer were shown to have a different elemental composition; more similar to soil samples than samples of the inner bone layer. PCA scores and loadings plots were preferred to dendrograms obtained using Cluster Analysis, due to the limits of the latter one to appreciate the spatial ordering of samples. Partial least squares discriminant analysis (PLS-DA), a frequently used supervised classification method, was applied to differentiate between degradation states of bone samples. PLS-DA results obtained in this study confirmed that changes derived from different burning conditions were associated with transformations in the mineral part of the bones. Accordingly, carbonized bones can be differentiated from cremated bones. Class assignment of bone samples with uncertain thermal conditions in dependence on their elemental composition has shown to be feasible. Consequently, for biochemical-archaeological studies the analysis and statistical classification of carbonized and cremated archaeological bones, as well as those exposed to unknown thermal conditions together with experiments in modern bones, are recommended.     

Restrictions on fluorine depth profiling for exposure age dating in archaeological bones

Archaeological fragments of bone and teeth that are exposed to a humid environment take up fluorine from the surrounding soil. The fluorine ion replaces the hydroxyl group in the mineral phase of the bone, forming chemically more stable fluorapatite. In cortical parts of the long bone diaphysis a fluorine concentration profile can be observed, which decreases from the outer surface and the marrow cavity towards the inner parts of the bone matrix. Geological time spans are needed for this process to reach equilibrium and for the distribution to become uniform. As the shape of the profile, which can be described by a diffusion model, contains information on the exposure duration of the fossil object, several attempts to use fluorine profiling as a dating method have been undertaken. The distribution of fluorine in an archaeological sample however is strongly influenced by environmentally induced processes of bone diagenesis, i.e. alteration in the structure and composition of the mineral phase and degradation of organic components that may make the time information indistinct. The primary chemical composition of bones can thus be obscured by diagenesis within tens, hundreds or thousands of years. This depends more on the diagenetic environment than on the geological age. To observe the impact of environmental influence on the profile shape, samples from several burial sites featuring various soil conditions have been analyzed for their fluorine distribution and preservation state. This paper provides an overview on the restrictions that have to be considered when attempts are undertaken to relate a fluorine diffusion pattern to the archaeological age of a bone specimen.     

Deficiencies and challenges in the study of ancient tuberculosis DNA

We explore the standards of research and reporting needed to justify the destructive analysis of archaeological human bone for biomolecular studies of ancient tuberculosis (TB). Acceptable standards in osteological interpretation have been met in some biomolecular papers, but there are also cases where insufficient care has been taken in distinguishing between pathognomonic lesions and those that are ‘consistent with’ a diagnosis of TB. Some biomolecular studies have failed to recognize that archaeological bones might be contaminated with environmental mycobacteria whose DNA could give rise to false positives in polymerase chain reactions directed at members of the Mycobacterium tuberculosis complex. The difficulties of applying spoligotyping to ancient DNA have also been underestimated and conclusions drawn from such analyses are often weakly supported. Assumptions that mycobacterial DNA preserves better than human DNA, and that contamination with modern DNA is less of a problem, has led in some cases to a laxity in research standards with insufficient attention paid to the need to authenticate ancient DNA results. We illustrate our concerns by reference to a recent paper reporting biomolecular detection of ancient TB DNA in skeletons from the eastern Mediterranean Neolithic settlement of Atlit-Yam. We are unconvinced that the skeletal evidence presented in this paper gives sufficient indication of TB to warrant destructive analysis, and we are concerned that during the biomolecular part of the project inadequate attention was paid to the possibility that results might be due to laboratory cross-contamination or to amplification of environmental mycobacterial DNA present in the bones.     

BONE POROSITY AND THE USE OF MERCURY INTRUSION POROSIMETRY IN BONE DIAGENESIS STUDIES*

Porosity measurements made on archaeological bones have revealed very-close relationships between changes in the porosity, remaining protein content and mineral alterations. The results have important implications for models that attempt to quantify the rates and extent of chemical reaction between bone and its geochemical environment. We report here on a novel application of an established technique, mercury intrusion porosimetiy, to investigate in more detail the pore size distribution of archaeological bones. Mercury intrusion porosimetry measures an ‘intermediate’range of bone porosity, ‘mesoporosity’, and produces data which permit the observation of significant structure characteristics in the porosity of modern laboratory altered and diagenetically altered bones.     

Destruction of microstructure in archaeological bone: a case study from Portugal

Sampling of archaeological human bone may not be justified, contrary to former high expectations regarding adult age assessment based on histomorphometry. The alterations in buried bone as a result of bacterial action are readily visible in the scanning electron microscope (SEM). An understanding of the chemical and structural changes to cortical bone requires work at the level of a few microns. This paper reports on problems encountered during analyses of samples of human bone from Mesolithic (ca. 8000 calbp) shell midden sites at Muge in central Portugal, and the methods used to try and overcome these problems. We believe we have shown that these Mesolithic bones are partly comprised of bacterially reprecipitated mineral, which has had collagen removed, with consequent obliteration of bone microstructure. We conclude that microbial destruction of the structure of archaeological bone can be a serious impediment to analysis of the characteristics of the population represented by those skeletal remains. Copyright © 2001 John Wiley & Sons, Ltd.     

The long–term survival of bone: the role of bioerosion

Fossil bones (N = 350) spanning more than 350 million years, and covering a wide range of depositional environments, were studied to compare the distribution of microbial destruction features in fossil bones with previously published data sets of bones of archaeological age. The distribution of bioerosion in fossil bones is very different from that found in bone from archaeological sites. Fossil bones typically show little or no bioerosion. Under normal conditions, if a bone is to survive into the fossil record, then rapid bioerosion must be prevented (or halted). This conclusion suggests that early post mortem processes,such as the mode of death, influence the potential of any bone to survive into deep time.     

The Red Fox (Vulpes vulpes) as an Accumulator of Bones in Cave‐like Environments

Identifying the behavioural patterns of bone collecting animals is a crucial aspect of taphonomic studies. Although many studies have established criteria for identifying animal‐collected or animal‐modified bones, very few papers describe the distinguishing features of fox‐made bone assemblages. The bone assemblage collected in an inactive underground stone mine in Potok‐Senderki (Poland) is diagnostic of a red fox (Vulpes vulpes) den. This site provides an ideal opportunity to develop an understanding of the bone collecting behaviour of red foxes in cave‐like environments. This study showed that bones collected by red foxes are concentrated in clusters. The bones represent a broad spectrum of local fox prey species, with most bones showing the marks of gnawing. Each cluster may contain from <10 to >100 bones. Furthermore, the long axes of the bones in clusters frequently show specific orientation. The analysis of bones at this site might make an important contribution towards the establishment of baseline criteria for the identification and evaluation of fox‐accumulated bone assemblages. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of chemical changes in bone material from South African fossil hominid deposits

The bone fragments of the Australopithecus Africanus from the dolomitic cave in the Cradle of Humankind in South Africa have been studied by the use of several spectral techniques. The aim was to establish their degree of preservation and possibilities of inferring the life conditions from them. X-ray diffraction studies revealed the transformation of the mineral components partially into fluoroapatite form with addition of goethite, birnessite and quartz phases and with surprisingly well preserved collagen remains. Several important chemical elements were detected by using the electron (EPMA) and synchrotron-based X-ray fluorescence (XRF) microprobes. Among them, Sr and Zn were distributed in a way following the distribution of Ca, the main element of the bones. We suspect the immanent crystallographic substitution of Ca in this case in native bone. Iron followed the distribution of Sr but while Sr is distributed in a continuous way in more rigid locations, iron occurs mainly in the edge zones of the bones and in spot-like inclusions inside. Some part of the spots located in a very edge of bone is also filled with Mn and Cu. It suggests that the porosity and fracture of bones plays a more significant role in the localization of Fe, Mn and Cu. They also form a rigid thin layer (100–250 μm) avoiding further penetration. As (as AsO₄³⁻?) distribution pattern is unique and seems to occur in those locations where the concentrations of Sr are small. The conditions for bone penetration as determined from Eh-pH diagrams suggest that is rather impossible to preserve the organic matter in locations of Mn and also that Mn and Fe hardly can meet in the same spots.     

The strange case of Apigliano: early ‘fossilization’ of medieval bone in southern Italy

The preservation of the osteological material at the medieval site of Apigliano, in southern Italy, is characterized by bones with highly crystalline, and altered, mineral phases. In addition to this, some material retains perfect histological preservation, with the exception of small microfissures present throughout this structure. Diagenetic porosity is indicative of collagen loss via chemical degradation. The levels of residual collagen in these bones are much lower than is predicted from simple models of gelatinization, and thus a more complex explanation for the state of preservation must be sought. Possible explanations for the rapid loss of bone collagen are considered, including a high–temperature event, the acceleration of hydrolysis due to liming and extreme wetting and drying cycles.     

Baboon Bone Mineral Densities: Implications for the Taphonomy of Primate Skeletons in South African Cave Sites

C. K. Brain (e.g., 1981) documented an interesting difference in relative skeletal part representation between primates and bovids of similar live body size recovered from the fossil cave site of Swartkrans (South Africa). Hominids and baboons are represented primarily by skull parts and a paucity of postcranial bones, while small sized bovids (Size Classes 1 & 2) are represented by more equivalent abundances of all skeletal elements. Brain argued that the Swartkrans bones were primarily carnivore collected, and postulated that the difference in element frequencies between primates and bovids is the manifestation of less durable primate postcranial skeletons relative to those of bovids when either are subjected to a destructive process such as carnivore feeding. Experiments by Brain (1981) in which baboon and bovid carcasses were fed to large, African carnivores lend support to this hypothesis. Bovid postcranial elements survived carnivore feeding more frequently and more completely than those of primates. Assuming that bulk bone mineral density (bulk BMD) is a measure of durability, we supplement Brain's observational data by presenting the first systematic, element-by-element comparison of baboon and bovid postcranial bulk BMD measurements obtained by dual energy X-ray absorptiometry (DXA). While the bovid in our sample exhibits greater absolute bulk BMD values in 57·8% of all measured bone areas, when grouping areas by body region, a statistically significant difference in absolute bulk BMD values between the baboons and bovids is documented only in the hindlimb region (i.e., femur, tibia and patella). Density differences in other body regions are not statistically significant. Further, in only one case (i.e., small bovids in the Member 2 [1948–1953/1965–1975 excavation] assemblage) is there a significant and positive correlation between skeletal part frequencies of primates or bovids and bulk BMD. This suggests that, for the most part, primate and bovid skeletal part representation at Swartkrans is not the result of density-mediated processes. Differences in skeletal part representation between primates and bovids at Swartkrans may thus be attributable to factors other than bone density—such as bone size, length, shape, and/or the relative palatability of surrounding soft tissues on bones.     

Micro-sampling of human bones for mobility studies: diagenetic impacts and potentials for elemental and isotopic research

The nature of long bone formation and the pathways of interaction between bone samples and the burial environment suggest that portions of the bones disconnected from the arterial system are resistant to diagenetic alteration. Preliminary work on femurs from Early Bronze Age hunter-gatherers in Cis-Baikal, Siberia shows that the nature and progression of chemical changes in the bone matrix due to microbial attack can be analyzed using laser ablation inductively coupled plasma mass spectrometry. Intra-osteon variability in elemental concentrations and strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) indicate the presence of unaltered portions of bone within diagenetically modified bone and suggest that useful data remain accessible. These biogenic signals can potentially be useful for mobility research in broad terms and the smaller timescales within an individual's lifetime (months, years), accessible therein. Laser ablation micro-sampling of femur specimens showed that intra-osteon elemental composition of Ba, Re, and Cs varied within and was correlated between multiple osteons of a single bone. Portions of chemically unaffected bone were identified within, and effectively discriminated from diagenetically altered bone tissue. Areas showing visual alterations and erratic or uncorrelated Ca and Sr elemental results also had anomalous Sr isotope ratios, suggesting diagenetic alteration in those places. Compositional and isotopic analysis of intact portions of bone supports the hypothesis that hunter-gatherer groups in Cis-Baikal made numerous major movements during their lives. Microscopic analysis of long bones clarifies aspects of biodeterioration and correlations between trace elemental results and diagenetic alteration. Micro-sampling of intact portions of bone expands the scope of available materials for research on mobility and other aspects of human past behavior.