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.     

Diagenesis and survival of osteocalcin in archaeological bone

It has been demonstrated that the protein osteocalcin can survive in bone in the archaeological record, and postulated that it has the potential to survive over geological time periods. The precise mechanism for this longevity of survival is not yet fully understood, and has not been extensively studied in comparison to other diagenetic aspects of archaeological bone. We report a comparison between osteocalcin survival and the state of preservation of more than 60 bones from 14 archaeological sites. The amount of osteocalcin, assayed immunologically, was compared with diagenetic parameters that measure: the amount of ‘collagen’ in the bone, the mineral changes, the porosity, and the histological preservation of the material. The findings indicate that microbial taphonomy and mineral alteration of bone have a profoundly damaging effect on the preservation of osteocalcin.     

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.     

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.     

Effects of Sodium Hydroxide Treatment and Ultrafiltration on the Removal of Humic Contaminants from Archaeological Bone

This study compared bone collagen extraction techniques that included treatment with sodium hydroxide and 30 kDa ultrafilters using a set of well‐preserved, humic‐contaminated archaeological marine mammal bones. Treatment with sodium hydroxide was effective at removing humic contaminants from archaeological bone, although yields were significantly decreased. Yields were also significantly decreased by ultrafiltration although this study produced no evidence that 30 kDa ultrafilters were effective at selectively removing humic contaminants from archaeological bone. The combination of sodium hydroxide treatment and ultrafiltration did not produce superior results to the treatment involving only sodium hydroxide. Archaeological samples exhibiting darker colouration indicative of humic contamination should be treated with sodium hydroxide to remove these contaminants. Copyright © 2017 John Wiley & Sons, Ltd.     

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.     

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.     

TESTING AN ALTERNATIVE HIGH‐THROUGHPUT TOOL FOR INVESTIGATING BONE DIAGENESIS: FTIR IN ATTENUATED TOTAL REFLECTION (ATR) MODE*

Archaeological bone undergoes alterations after burial (diagenesis) that constitute a problem for the survival of archaeological information. A common method to assess this alteration is Fourier transform infrared spectrometry (FTIR). However, the commonly applied method (FTIR–KBr) is destructive and sample preparation may influence the results. This paper tests the suitability of FTIR attenuated total reflection (FTIR–ATR), a method not commonly used to investigate bone diagenesis. FTIR–ATR requires less sample preparation and can be non‐destructive, allowing analysis of bone cross‐sections. Modern and archaeological bones were analysed using both methods and different sample preparation methods were tested. The results show that FTIR–ATR has advantages for the rapid assessment of bone 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.     

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.     

Endocranial lesions in non‐adult skeletons: understanding their aetiology

Reactive new bone on the endocranial surface of the skull in non‐adults has recently received a lot of attention in the palaeopathological literature. These features appear as layers of new bone on the original cortical surface, expanding around meningeal vessels, as isolated plaques, ‘hair‐on‐end’ extensions of the diploë or, as ‘capillary’ impressions extending into the inner lamina of the cranium. These lesions are commonly found on the occipital bone, outlining the cruciate eminence, but have also been recorded on the parietal and frontal bones, and appear to follow the areas of venous drainage. Although recognized as resulting from haemorrhage or inflammation, their precise aetiology is still a matter of controversy. This paper outlines their possible causes and examines their nature and distribution in a group of non‐adults from four archaeological sites in England. It is recommended that, when recording these lesions in the future, additional skeletal pathologies, the age of the child, and nature and distribution of the lesions also be taken into account. Copyright © 2004 John Wiley & Sons, Ltd.     

Stable nitrogen isotope values in arid‐land kangaroos correlated with mean annual rainfall: potential as a palaeoclimatic indicator

Bone collagen stable nitrogen isotope values are reported for modern kangaroos (Macropus spp.) from eight field sites along a 1160 km south–north transect from temperate coastal to arid interior South Australia, in order to investigate δ¹⁵N tissue variability in relation to rainfall and relative humidity. Mean annual rainfall along the transect ranges from 775 mm at Mount Gambier in the southeastern coast to 176 mm at Innamincka in the arid northern interior, while 3pm relative humidity ranges from 73% at coastal Flinders Chase to 36% at Innamincka. In arid habitats (176–238 mm rainfall), the δ¹⁵N values of kangaroo bone collagen become more positive in relation to decreasing mean annual rainfall (r² = 0.98), while there is only a weak correlation with relative humidity (r² = 0.67). In contrast, in temperate and semi‐arid coastal habitats (350–775 mm), there is no correlation between kangaroo bone collagen δ¹⁵N values and rainfall (r² = 0.0011) or between δ¹⁵N values and relative humidity (r² = 0.0035). Thus, in South Australia, kangaroo bone collagen δ¹⁵N values do not show a simple linear correlation with either rainfall or relative humidity across all habitats. These results suggest that stable nitrogen isotope analyses of herbivore bones obtained from archaeological and palaeontological sites may provide a reliable proxy for past rainfall values in arid‐land ecosystems. Copyright © 2007 John Wiley & Sons, Ltd.     

Well preserved non‐collagenous extracellular matrix proteins in ancient human bone and teeth

Ancient bones in a good preservation state, ascertained by microscopic techniques, conserve extracellular matrix proteins over thousands of years. With new techniques, intact extracellular matrix proteins from ancient bones and teeth are extracted and separated by one‐dimensional and two‐dimensional electrophoresis. Proteins were identified in Western blots by special antibodies against different human extracellular matrix (ECM) molecules of bone. We have confirmed different types of ECM human bone molecules such as osteonectin, osteopontin, and alkaline phosphatase with specific antibodies in human bone samples from different age groups. Additionally, we selected bone samples from different cultural time periods, such as the Middle Ages, the Bronze Age and the Late Pre‐Pottery Neolithic Phase (PPNB), and teeth from individuals from the Early Middle Ages and from the Late PPNB. The survival of intact extracellular matrix proteins in ancient bones and teeth dating from recent times to the Late PPNB, and reliable techniques to identify these proteins, present a great challenge to further research. A Match Set with PD‐Quest 7.2 shows that only 16% of protein spots in the teeth are also found in the bone of the same individual. In combination with the results of macro‐ and microscopic investigation, biochemical techniques will help us in obtaining a better understanding of bone and teeth in health and disease. Copyright © 2006 John Wiley & Sons, Ltd.     

Sexual dimorphism in diaphyseal cross‐sectional shape in the medieval Muslim population of Écija,Spain, and Anglo‐Saxon Great Chesterford,UK

Differences in adult male and female activity patterns may influence levels of sexual dimorphism in physical dimensions, including the cross‐sectional shape of long bone diaphyses. Previous studies of archaeological populations have demonstrated significant differences in diaphyseal shape between males and females. In this study, dimorphism in external diaphyseal shape of upper and lower limb bones (reflected in indices of external diaphyseal diameters), and bilateral asymmetry in these indices, were examined in two medieval populations: Muslim Écija (Spain) and Anglo‐Saxon Great Chesterford (UK). Attempts were made to relate observed patterns to documentary and other osteological evidence for differences in male and female activity patterns. While few significant differences in upper limb bone cross‐sectional shape were observed in either population, significant differences in shape were found in the lower limb diaphyses at Écija at the femoral midshaft and tibial foramen and midshaft levels, and at the tibial midshaft for Great Chesterford. Comparison with published data suggests that these differences are marked for Écija, and perhaps fairly high for Great Chesterford compared with other populations with an agriculture‐based economy. This is consistent with documentary and osteological evidence suggesting marked gender differences in behaviour in medieval Muslim Spain. No significant differences in bilateral asymmetry were found, but the effects of small sample size cannot be ruled out. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Histological identification of bone fragments in archaeology: telling humans apart from horses and cattle

In archaeology, it is not always possible to identify bone fragments. A novel approach was chosen to assess the potential of histology as an identification tool in those instances in which macroscopical study (e.g. by means of bone surface texture) has failed. This study concentrated on the diaphyses of long bones in three species of comparable size which are relevant to archaeology. Late juvenile and adult human diaphyseal bone structure was compared with the bone structure of horses and cattle. Since bone structure can differ even within a single bone, the restrictions in terms of bone category, bone part and species were deemed necessary for the development of a useful identification method for archaeological bone. The reference series comprised long bones from several individuals to broaden the insight into variations in diaphyseal bone structure within a single species. A general difference in the primary bone types was found between humans and the two large mammals. Human bone displayed lamellar bone types, whereas horses and cattle showed fibrous bone types. The only exceptions were separated growth layers. A difference in the secondary bone structure was also observed. A large number of connecting, primary (Volkmann's) canals, giving the secondary bone a reticular aspect, were common in horses and cattle. They were not, however, present in the human bones studied. To confirm the validity and applicability of these differences, a blind test was conducted on 15 diaphyseal fragments of identified long bones from archaeological sites. The results were very promising. Although four fragments could not be identified due to severe degradation, all the others were correctly attributed. Copyright © 2006 John Wiley & Sons, Ltd.     

Palaeo‐otology of cholesteatoma

A cholesteatoma is a collection of dead epithelial cells in the ear which becomes infected with low‐grade pathogenic microorganisms. The lesion tends to cause resorption of the surrounding bone, which potentially permits its recognition in skeletal remains. Cholesteatoma may occur in the middle or the outer ear. Using two examples of cholesteatoma in skeletal remains from British archaeological sites, the differential diagnosis of cholesteatoma from other aural diseases is demonstrated. Some of the differences in bony lesions caused by middle ear and external ear canal cholesteatoma which may enable the two to be distinguished in palaeopathological specimens are discussed. Attention is drawn towards the importance of making this distinction in ancient skeletal remains; middle ear and external ear canal cholesteatoma have a different array of causes and very different impacts upon health status. Copyright © 2005 John Wiley & Sons, Ltd.     

A taphonomic study of bone modification and of tooth‐mark patterns on long limb bone portions by suids

This work presents new taphonomic data on bone modification by suids, including domestic pig, wild and hybrid boars. The intense modification undergone by bones from animals smaller than 100 kg is shown, together with a more moderate modification on bones from larger animals. Both the ravaging pattern (with preferential deletion of cancellous tissue) and the tooth‐marking frequencies are similar to those documented among hyenas and dogs when having primary access to complete bones. A dual‐patterned experimental model consisting of the interaction of humans and suids was also considered. Here it is shown how suid modification of hammerstone‐broken bone assemblages is different from that documented among canids and hyaenids, as experimentally replicated. These results increase the number of non‐anthropogenic bone‐modifying agents and posit new issues on equifinality processes. Copyright © 2008 John Wiley & Sons, Ltd.     

Experimental chemical degradation compared to natural diagenetic alteration of collagen: implications for collagen quality indicators for stable isotope analysis

Stable isotopic ratios from archaeological bone collagen are valid palaeodietary indicators, but depend on sufficiently well preserved molecules and several collagen quality criteria have to be fulfilled (mostly collagen wt%, C%; N%, C/N molar ratio). For a reassessment of these quality criteria, and a better understanding of the chemical degradation of bone collagen, experimentally degraded modern bones and 54 archaeological human bones were investigated. In the course of the experimental degradation, alterations of isotopic ratios were paralleled by altered collagen quality criteria. The contrary was found in the case of the archaeological specimens. This implies that the commonly used collagen quality criteria may be insufficient and do not guarantee that stable isotopic values of the gelatine extracts will still represent the original biological signal.     

Uranium and Vanadium Concentrations as a Trace Element Method for Identifying Diagenetically Altered Bone in the Inorganic Phase

Archaeologists have generally avoided analyzing inorganic isotopes in bone because of its high porosity, large crystalline lattice spacing, and small crystallite size, making it particularly susceptible to diagenetic alteration. Because the inorganic isotopes are left unstudied, we lose a significant portion of information pertaining to an individual’s life history, such as migration, health, and ranging behavior. Tooth enamel, which does not have the same susceptibility to diagenesis as bone, can be used to extract this information but this means that taxa lacking teeth, such as birds, some species of fish, and some reptiles, are excluded. Here, we present a method that can be used to identify diagenetic alternation in bone. This is done by focusing on abnormal concentrations of vanadium and uranium. Neither element is readily bio-precipitated into hydroxyapatite due to ionic radius, vibrational frequency, atomic mass, and ionic charge. This makes them an ideal marker for diagenetically altered bone. Vanadium occurs in very low concentrations in modern bone, while archaeological bone shows clear evidence of normal, non-diagenetically altered values alongside high concentrations of vanadium in diagenetically altered bone. Uranium also is a measure of diagenetic alteration, as modern bone has concentrations below detectable limits (0.017 ppb), while some archaeological bone contains uranium above detectable limits. The biogeochemistry of these elements in soil and bone are discussed with implications for enamel studies.