Manual for the Preparation and Staining of Embedded Natural Dry Bone Tissue Sections for Microscopy

In the last decade, the use of light microscopy has been firmly established for the investigation of exhumated human bone tissue. As a rule, these remains cannot be decalcified; thus, they are most commonly prepared for microscopic analysis as ground thin sections. These ground sections are of great value in diagnosing disease, in estimating age or in assessing taphonomic alteration. As bone is sometimes fragile and can be damaged by the grinding process, the specimen is occasionally supported by an embedding medium. In contrast to the vast amount of research conducted on embedded and unembedded unstained bone material, the use of histological stains on undecalcified dry bone tissue has been long neglected. In this article, a new method for embedding, sawing and grinding dry bone tissue is presented. The produced sections are subsequently stained with haematoxylin. The results show that even ground sections of fragile bone can be made in a quick and easy manner. Staining these sections enhances the envisioning of micro‐architecture and taphonomical processes. In addition, the sections stay open for inspection under polarized light. The results were consistent throughout the used bone material. To keep the method as accessible and comprehensive as possible, a step‐wise manual is provided. An additional troubleshooting paragraph discusses the most often encountered problems and provides solutions. Copyright © 2011 John Wiley & Sons, Ltd.     

The use of dental criteria for estimating postnatal survival in skeletal remains of infants

Separate cemeteries and/or burial loci for infants have been variously interpreted as reflecting segregation by age, infanticide or even child sacrifice. Attempts to distinguish between these factors rely primarily on the age distribution found. Currently long bone length is the most commonly used method for fetuses and infants in the perinatal period, but its accuracy is affected by the inherent variation in size for age. We show here how to distinguish between death in the perinatal period and that occurring later in infancy through identification of the neonatal line in ground sections of deciduous teeth. The methodology is reviewed and applied to validate estimations of postnatal survival for infant remains recovered from two archaeological sites in Israel.     

Different Preparation Techniques – Similar Results? On the Quality of Thin‐Ground Sections of Archaeological Bone

Palaeohistology as a valuable diagnostic instrument is dependent on the production of high‐quality thin‐ground sections from dry bone. The objective of this study was to consider technical differences and assess the qualitative outcomes of five techniques for preparing thin‐ground sections from dry archaeological bone. Established techniques with long follow‐up times and excellently documented results were compared with simpler and cheaper time‐saving techniques. Evaluations were made of the quality of thin sections obtained by one classical machine‐based embedding technique, two revised versions of the same technique, one manual moulding technique based on Frost's rapid technique and one manual hybrid technique. Five osteological specimens of differing quality were prepared following the manuals for these five techniques and examined microscopically with respect to a list of standardised histological and diagenetic parameters. Alterations in the specimens attributable to preparation effects were recorded, and observations were scored with reference to three criteria: section quality, technical quality and staining. The results show that embedding techniques are to prefer. Superglue should not be used as a mounting or embedding medium. Manual grinding comes with several limitations, and machine cutting and grinding are preferred. Haematoxylin staining can be successfully applied to embedded specimens, giving more information on microscopic diagenetic processes. A stepwise manual for a revision of the classical embedding technique is presented. The time required for producing sections using classical embedding techniques is shortened from 6 weeks to 3.7 days by refining the preparation/polymerization processes involved with no loss of osteological data. Copyright © 2014 John Wiley & Sons, Ltd.     

Bone preservation in human remains from the Terme del Sarno at Pompeii using light microscopy and scanning electron microscopy

Archaeological bone can show marked and complex alterations depending on the environment in which it was buried. In this study, the state of preservation of 27 femurs recovered from the archaeological site of Pompeii was evaluated by light microscopy and scanning electron microscopy. Most of the bone samples, prepared by the grinding method, showed good histological preservation, although they were characterized by microfissures (microcracking). Nine bone samples showed different states of histological preservation, including worst preservation (two femurs), due to diagenetic processes. Cryostat bone sections stained with thionin or 4′,6′-diamidino-phenylindole (DAPI) revealed the persistence of DNA within some osteocyte lacunae. Scanning electron microscopy analysis showed that ultrastructural characters, such as lamellae and collagen fibres, are recognizable only in unaltered bone. Our results reveal that most Pompeian samples are well preserved since they have a bone microstructure virtually indistinguishable from that of fresh bone. In methodological terms, although each of the various morphological methods used contributes information, histological and histochemical analyses are the most informative for studying the preservation state of bone and allow for rapid essential screening of archaeological bone.     

Observatory validation of Neolithic tells (“Magoules”) in the Thessalian plain,central Greece,using hyperspectral spectroradiometric data

This paper presents the results obtained from field spectroradiometric campaigns over Neolithic tells (“magoules”) located at the Thessalian region in Greece. In each one of the four archaeological sites selected, three sections were carried out using the GER 1500 handheld spectroradiometer. Spectral profiles of the sections and several vegetation indices such as Normalized Difference Vegetation Indices (NDVI) and Simple Ratio (SR) have been examined in this study. This is one of the first times that ground hyperspectral data are evaluated in such context of archaeological research for the spectral characterization of archaeological features. As it was found, ground spectroradiometric measurements can be efficiently used in order to support and validate satellite imagery results for the detection of archaeological sites.     

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.     

Scanning electron microscope study of normal vertebrae and ribs from early medieval human skeletons

A method is described for the preparation of vertebral body and rib sections of archaeological context, for scanning electron microscopy (SEM). The specimens included in the present paper are macroscopically normal and are male and female in juvenile and adult age brackets. The changing directionality of trabeculae and the changing ratio of trabecular bone to cancellous space are noted in relation to the age at death.     

Biofilm growth in human skeletal material from ancient Mesopotamia

Investigators have long recognised the effects of microbial activity on archaeological bone. These investigators, however, have focused on single or groups of microbes rather than on complex microbial aggregates such as biofilms, a focus that has affected our understanding of archaeological bone biodeterioration. In this paper, we report on the investigation of a biofilm in archaeological human bone from the site of Tell Leilan, Syria (2900–1900 BCE). Scanning electron microscopy indicated that the biofilm is characterised by single cells and microcolonies of bacteria and fungi, as well as calcite crystals that were all embedded within extracellular polymeric substances. Using culture techniques and DNA sequencing, we isolated and identified several microbes from the biofilm including Amycolatopsis sp., Penicillium chrysogenum, Aspergillus sp., Chaetomium sp., and Cladosporium sp. Having characterised the Leilan biofilm, we are now closer to understanding the complex process of bone biodeterioration in archaeological bone collections.     

An Investigation Into the Relationship Between Funerary Treatment and Bacterial Bioerosion in European Archaeological Human Bone

A central problem in funerary archaeology is interpreting how the corpse was manipulated in the immediate post mortem period. The extent of bacterial bioerosion to the internal bone microstructure has been proposed as a means to infer the early post mortem history of a corpse, as it has been suggested that this form of bone diagenesis is produced by an organism's putrefactive gut bacteria. Under this model, different forms of funerary treatment would be expected to leave characteristic signatures of bioerosion in archaeological bone. Here, we tested the extent to which bacterial bioerosion of ancient human bones reflected funerary treatment, through histological analysis of 301 archaeological human bone thin sections from 25 European archaeological sites. We found that bioerosion was significantly influenced by whether a bone originated from a neonatal individual or an anoxic context. When these remains were excluded, bioerosion was controlled by archaeological phase in a manner consistent with known early post mortem treatment and forensic models of bodily decomposition. These findings suggest that microscopic analyses of bone have useful applications in reconstructions of funerary processes and provide some insight into factors that may control the persistence of organic biomolecules and fossilization.     

A distinct region of microarchitectural variation in femoral compact bone: Histomorphology of the endosteal lamellar pocket

Bone is a dynamic tissue, responding locally to differential mechanical loading and systemically to hormonal stimuli. Although the tendency is to consider compact bone a homogenous tissue, regional variations in microarchitecture are known to have quite different properties relating to processes of growth and mechanical loading. Specifically, we investigated an example of microstructural variation of the human, adult femoral endocortex, referred to here as the endosteal lamellar pocket (ELP). The femoral ELP is characterised as a histomorphological meta‐feature, made distinct from general circumferential lamellae by its hemicircumferential lamellar orientation, medial positioning, radially oriented Volkmann's canals and decreased presence of Haversian systems. Our preliminary observations of mid‐diaphyseal transverse thin‐ground sections demonstrate the ELP as a regular meta‐feature of adult human femora unearthed from the Maya archaeological site of Xcambó, Mexico. ELPs were easily identified in 38 of 45 examined individuals. Their size, shape and degree of secondary remodelling were variable. In comparison, ELP lamellae and those originating from the periosteum are divergent, or non‐concentric in orientation. This divergence, along with the ELP's medial position, suggest it forms during growth through endosteal modelling drift. Possible considerations for ELP formation and persistence with age are discussed as well as its implications for the study of bone dynamics during growth and activity. Further research is necessary to quantitatively measure the morphology of ELPs and to identify any co‐variance between ELP characteristics and age, sex or indicators of mechanical loading. Copyright © 2010 John Wiley & Sons, Ltd.     

Confocal laser scanning microscopy: a flexible tool for simultaneous polarization and three-dimensional fluorescence imaging of archaeological compact bone

Wide-field polarized light and epifluorescence microscopy have been used to enhance analysis of archaeological bone tissue, providing information on bone formation, modeling, pathology, preservation, age estimation, and biomechanics. Though valuable, these techniques are limited by their inability to remove out-of-focus light and view multiple levels of a sample, restricting our understanding of the three-dimensional (3-D) microarchitecture of compact bone. Modern technological advances, such as microscopic computerized tomography, allow increasing resolution in 3-D bone imaging, but do not allow fluorescence labeling or polarized-light analysis. Confocal laser scanning microscopy (CLSM) is a valuable tool for 3-D histology. However, its application to the study of compact bone is lacking, especially in archaeological and forensic sciences. The current study investigated CLSM as a tool for fluorescence and polarized-light microscopy of archaeological compact bone in order to demonstrate its advantages. Standard techniques and CLSM are compared in their suitability for imaging well preserved archaeological bones from the Dakhleh Oasis, Egypt. CLSM's high resolution, multi-channel, two- and three-dimensional capabilities augment the flexibility and creativity of compact bone imaging and have the potential to increase the accuracy of quantitative medical and anthropological histomorphometric techniques. CLSM is specifically suggested as a useful tool for the investigation of ancient bone fluorescence caused by the presence of tetracycline and/or other fluorochromes.     

EVALUATION OF BINDER/AGGREGATE RATIOS IN ARCHAEOLOGICAL LIME MORTARS WITH CARBONATE AGGREGATE: A COMPARATIVE ASSESSMENT OF CHEMICAL,MECHANICAL AND MICROSCOPIC APPROACHES*

This paper reports on the comparative evaluation of traditional and innovative methods for the investigation of mortar technology. Preliminary results of various approaches to determining the mix proportions in lime mortars with carbonate aggregate are presented. Quantitative analysis was tested on ad hoc prepared mortars of known composition containing carbonate aggregates of different origin (travertine and Bath limestone). Wet chemical methods, manual disaggregation and Digital Image Analysis of thin sections and cross‐sections stained with Alizarin Red S were tested. The advantages and drawbacks of the different approaches are discussed, and applications to the analysis of archaeological mortars and plasters are highlighted.     

The taphonomy of cooked bone: characterizing boiling and its physico–chemical effects

Cooking is perhaps the most common pre–burial taphonomic transformation that occurs to bone, yet it is still one of the least understood. Little progress has been made in determining a method of identifying cooked bone in the archaeological record, despite its import for various branches of archaeology. This paper attempts to describe boiling in terms of its physico–chemical effects on bone, and uses a suite of diagenetic indicators to do this. It is shown that cooking for brief periods of time has little distinguishable effect on bone in the short term, but that increased boiling times can mirror diagenetic effects observed in archaeological bone. The relationship between the loss of collagen and alterations to the bone mineral is explored through heating experiments, and the results compared with archaeological data.The possibility of boiling being used as an analogue for bone diagenesis in future studies is raised, and the key relationship between protein and mineral is once again highlighted as vital to our understanding of bone diagenesis.     

Actualistic research into dynamic impact and its implications for understanding differential bone fragmentation and survivorship

The relationship between bone mineral density and archaeological bone survivorship has played a critical role in zooarchaeological and taphonomic studies in recent decades. Numerous studies have suggested that higher-density skeletal element portions survive more frequently than lower-density element portions when archaeological assemblages are affected by some taphonomic processes. Interpretations of density mediated destruction have become commonplace in the archaeological literature, and are often used to explain the absence of certain bone elements and element parts in zooarchaeological assemblages. This study explores the effects of rockfall on bovid elements in varied environmental conditions and the differential survivorship of their element parts, and has implications for understanding the taphonomic processes through which bones are subjected to dynamic loading. Actualistic rockfall experiments conducted on twelve samples of frozen, fresh, and semi-dried bovid bones reveal that the generally low-density epiphyseal ends of bone elements resist fracture and analytical deletion with more frequency than the higher-density diaphyses. This evidence suggests that bone density does not correlate with likelihood of breakage or effective archaeological “destruction” when rockfall and other processes that result in dynamic impact are in action. While this research does not question the relationship between bone mineral density and the likelihood for archaeological survivorship as the result of some taphonomic processes, it presents one specific set of taphonomic processes that result in the differential survivorship of low density bone elements parts and the fragmentation and destruction of higher density element parts. This research presents evidence that shows that dynamic impact is a process capable of fragmenting and sometimes destroying high-density elements while low-density elements survive.     

The bioerosion of bird bones

This study presents a synthesis of the evidence for modern archaeological, and palaeontological bioerosion of vertebrate tissue. It describes the first evidence for the bioerosion of modern, archaeological and fossil bird bones. A new form of bone bioerosion, known as Hackett tunnels, is defined. The bioerosion of vertebrate tissue by cyanobacteria and algae in modern marine and lacustrine environments is also described. Archaeological evidence indicates that the destruction of bone by bioerosion occurs in other terrestrial environments, such as cave deposits and middens. Bone from marine and lacustrine environments that appears to be macroscopically well preserved can have large amounts of microscopic fabric destruction. © 1997 John Wiley & Sons, Ltd.     

Probable persistence of the remains of a Rouget-Neumann sheath into the canalicular system of ancient human bone

Examining histological sections obtained from human bones from Iron Age archaeological excavations (about third to fifth century BC , Alfedena and Sulmona, central Italy), the authors show the presence of an amorphous, afibrillary substance inside the osseous canaliculus in decalcified sections. This substance has shown unusual chromaffinity to staining using the Schmorl method with thionine and phosphowolframic acid. It is probably formed by acid mucopolysaccharides obtained from a transitory betametachromasia with toluidine blue staining at neutral pH. This metachromasia persists after digestion in bacterial collagenesis, but disappears after digestion in bovine testicular jaluronidasis. These reactions indicate that the endocanalicular substance may represent well-kept remains of a Rouget-Neumann sheath, which surrounds cytoplasmic extensions of osteocytes.     

Microscopic examination of ground sections – a simple method for distinguishing between bone and antler?

Both bones and antlers have been used throughout history for manufacturing various products. Therefore, archaeologists often have to distinguish between these materials. The more the raw material (bone or antler) has been treated, the more complicated it is to distinguish. For this study, recent macerated and archaeological samples of bone and antler and their products have been compared. Antler is usually very easy to distinguish based on the characteristic surface structure; however, the original surface is not preserved in all artefacts. The inner surface of the diaphysis of long bones is mostly compact with prominent trabeculae and lamellae, whereas, after removal of the spongiosa, the inner surface of antler is characterised by numerous grooves. Sometimes, however, artefacts are found which are composed only of the compact bone substance, without inner and outer surfaces, and it is not possible to distinguish between bones and antlers macroscopically. Haversian systems can be found in histological and ground sections of both bones and antlers. In contrast to bones, antlers have few interstitial lamellae. The osteons of antler seem to be smaller and to consist of fewer layers than those of bone. Inner and outer circumferential lamellae cannot be distinguished in antlers. Although microscopic examination of sectioned samples does not allow distinction between bones and antlers in every case, it represents a valuable additional tool for distinguishing between these two materials in historic artefacts. Copyright © 2007 John Wiley & Sons, Ltd.     

Serum Proteins in Archaeological Human Bone

Research on the decomposition of bone collagen offers the key to a wealth of hidden information, for instance, to understanding palaeoclimatic conditions; habitat-specific parameters such as altitude; and for the reconstruction of palaeodiet and subsistence patterns. Radiocarbon dating, one of the most commonly used geochronological techniques is also preferentially carried out on bone collagen. Because negatively charged ions, and especially phosphate groups, are responsible for the tight bonding between organic molecules and bone, soluble serum proteins need not necessarily be leached from buried bone. The authors' hypothesis is that intruding minerals, aided by recrystallization of the bone mineral matrix, and colloid formation, aided by humic substances, form protective layers that preserve serum proteins in bone even after long periods of burial. We have used electrophoresis and Western blotting to recover protein fractions from about 150 archaeological bones from various sites and epochs (up to 5500 BC ) and have succeeded in recovering, purifying all, and identifying some of the proteins. Molecular weight bands corresponding to albumin, transferrin and a-2HS-glycoprotein (A2HS) were frequently recovered. This paper presents the method for separating serum proteins from archaeological bone and for their identification. Twenty-five per cent of samples with the respective molecular weight band still gave a positive immunological reaction with antibodies. We conclude that non-collagenous proteins, especially serum proteins, may be well preserved in bone.