What is histology?
The term histology is derived from the Greek words histos and logos meaning 'web or tissue' and 'speak or account'. It refers to the microscopic study of all animal and plant cells or tissues. For soft tissues these are normally embedded in a soft substrate such as paraffin wax before being cut into fine slices using a microtome. These slices may then be stained and examined using optical or transmission electron microscopy (TEM). Because bone is both much harder and more brittle than the body's soft tissues it is difficult to section with a microtome in the usual way. Specimens are either demineralised using mineral acids or a calcium chelating agent such as ethylenediamine-tetraacetic acid (EDTA) before sectioning in the same way as soft tissues or, alternatively, sawn sections of whole bone are ground and polished with abrasives to the required thickness for examination in reflected light or by scanning electron microscopy (SEM).
In light microscopy, a wide range of structures is visible in thin sections of whole bone, even without staining. In mature compact bone the most striking of these features are the numerous circular structures. At the centre of these features lies a canal that encloses blood vessels, nerves and loose connective tissue and around which bone cells are arranged in concentric layers. This whole complex of concentric lamellae of bone surrounding a Haversian canal is called a Haversian system or osteon. Haversian systems represent secondary bone tissue that has been deposited during remodelling of primary bone.
Primary bone tissue or woven bone is the first to form in bone growth and repair. This temporary structure is gradually replaced by resorption around blood vessels and subsequent deposition of secondary bone, leading to the characteristic form of osteons. Immature or woven bone often persists in the area of tendon insertions and ligaments although these themselves may become mineralized in old age. Primary bone tissue has an irregular arrangement of collagen fibres, a higher proportion of bone cells or osteocytes and a lower mineral content than the denser secondary bone. During growth then and throughout adult life, there is a continuous destruction and rebuilding of Haversian systems and this succession is often reflected in overlapping osteons. Primary bone can often still be distinguished in the outer and inner circumferential lamellae lying close to the external and internal surfaces of bones. This has led some texts to describe the structure of bone as having an inner and outer layer of lamellar bone, separated by Haversian bone. Haversian bone, however is generally restricted to the skeletons of larger mammals whose longer lifespans necessitates a more developed mechanism for bone maintenance and repair.
In light microscopy, a wide range of structures is visible in thin sections of whole bone, even without staining. In mature compact bone the most striking of these features are the numerous circular structures. At the centre of these features lies a canal that encloses blood vessels, nerves and loose connective tissue and around which bone cells are arranged in concentric layers. This whole complex of concentric lamellae of bone surrounding a Haversian canal is called a Haversian system or osteon. Haversian systems represent secondary bone tissue that has been deposited during remodelling of primary bone.
Primary bone tissue or woven bone is the first to form in bone growth and repair. This temporary structure is gradually replaced by resorption around blood vessels and subsequent deposition of secondary bone, leading to the characteristic form of osteons. Immature or woven bone often persists in the area of tendon insertions and ligaments although these themselves may become mineralized in old age. Primary bone tissue has an irregular arrangement of collagen fibres, a higher proportion of bone cells or osteocytes and a lower mineral content than the denser secondary bone. During growth then and throughout adult life, there is a continuous destruction and rebuilding of Haversian systems and this succession is often reflected in overlapping osteons. Primary bone can often still be distinguished in the outer and inner circumferential lamellae lying close to the external and internal surfaces of bones. This has led some texts to describe the structure of bone as having an inner and outer layer of lamellar bone, separated by Haversian bone. Haversian bone, however is generally restricted to the skeletons of larger mammals whose longer lifespans necessitates a more developed mechanism for bone maintenance and repair.
Samples and sample preparation
At a microscopic level, the structure of ancient bone can be examined in a number of different ways. Well-established techniques include optical microscopy (OM), microradiography (µR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). More recent developments in microscopy that see applications in the analysis of archaeological and fossil bones are confocal laser scanning microscopy (CLSM), near-field scanning optical microscopy (NSOM), atomic force microscopy (AFM) and X-ray microtomography (µCT), a development of medical computed axial tomography (CT).
Some of these latter techniques are highly specialised, requiring large and prohibitively expensive facilities, and are therefore restricted to exceptional specimens or studies that aim to test and refine methodologies. By far the most common techniques applied to archaeological specimens are optical and electron microscopies in their various forms.
The techniques used to prepare, section and image specimens of archaeological bones grew out of standard histological methodologies developed for looking at modern tissues, including both soft tissues and mineralized tissues. When examining samples of bone from biopsies, surgery or autopsy it is still common to decalcify the specimens to leave only the organic (collagen) matrix and its associated cells which can then be fixed, dehydrated, embedded in paraffin wax and sectioned. In fresh bone specimens it is necessary to first stabilise the cells and other soft tissues from putrefaction by “fixing” them with a cross-linking agent, normally a 10% solution of formaldehyde in phosphate buffered saline.
In archaeological specimens bacterial degradation and other diagenetic processes have normally left the bone devoid of all cellular materials, so there is no requirement to fix them. However, they are usually cracked and friable and it is necessary to impregnate them with a suitable resin before attempting to prepare any kind of section. This in turn influences the approach adopted in producing sections. The methodologies described below are suitable for the preparation of both thick sections for electron microscopy, etc., and thin sections for light microscopy. Processes marked with an asterisk apply to the preparation of thick sections. The diagram is reproduced courtesy of Wiley.
Some of these latter techniques are highly specialised, requiring large and prohibitively expensive facilities, and are therefore restricted to exceptional specimens or studies that aim to test and refine methodologies. By far the most common techniques applied to archaeological specimens are optical and electron microscopies in their various forms.
The techniques used to prepare, section and image specimens of archaeological bones grew out of standard histological methodologies developed for looking at modern tissues, including both soft tissues and mineralized tissues. When examining samples of bone from biopsies, surgery or autopsy it is still common to decalcify the specimens to leave only the organic (collagen) matrix and its associated cells which can then be fixed, dehydrated, embedded in paraffin wax and sectioned. In fresh bone specimens it is necessary to first stabilise the cells and other soft tissues from putrefaction by “fixing” them with a cross-linking agent, normally a 10% solution of formaldehyde in phosphate buffered saline.
In archaeological specimens bacterial degradation and other diagenetic processes have normally left the bone devoid of all cellular materials, so there is no requirement to fix them. However, they are usually cracked and friable and it is necessary to impregnate them with a suitable resin before attempting to prepare any kind of section. This in turn influences the approach adopted in producing sections. The methodologies described below are suitable for the preparation of both thick sections for electron microscopy, etc., and thin sections for light microscopy. Processes marked with an asterisk apply to the preparation of thick sections. The diagram is reproduced courtesy of Wiley.
Important ethical note
Many archaeologists and curators are understandably concerned about clumsy sampling of, and needless damage to, human or animal remains in their custody. The person doing the sampling should therefore aim to be methodical in their approach and strive to minimise risk of damage to any bone specimen. When sampling specific skeletal elements it is best to avoid critical features that may be used later by other researchers making morphometric or other measurements.
As always when dealing with human remains, due regard to ethical guidelines and local laws relating to the handling of human tissues must be adhered to.
As always when dealing with human remains, due regard to ethical guidelines and local laws relating to the handling of human tissues must be adhered to.