1.4.1 Introduction
In the previous section x-ray methods for the measurement of bone mass have been described ending in the method of studying trabecular patterns on radiographs which depends on both the amount and arrangement o f bone. For the remainder of the chapter the emphasis is on techniques that may impart structural information. Because the background of these methods is generally less well known in the osteoporosis field than x-ray based methods, more detailed descriptions are given.
Many studies have shown that a change in bone microstructure occurs with age. The observed significant increase in trabecular separation (Parisien et al 1988; Christiansen et al 1992) and the decrease in mean trabecular plate density (trabecular number) with age (Parfitt et al 1983) suggests a loss o f whole trabecular elements and a disintegration o f the trabecular network. These conclusions and resulting theories on the mechanism of bone loss, some of which have been discussed in Section 1.2.1, have all been reached on the basis of information derived from histomorphometry. This technique is regarded as the gold standard for assessment of trabecular microstructure. The bone biopsies that have formed the basis of much information about bone structure are usually taken from the iliac crest which is a superficial non weight-bearing part o f the skeleton.
Electron microscopy reveals sections o f trabecular bone as a complex three dimensional (3D) network of curved plates and bars. The size, shape, orientation, distribution and connectivity of these structural elements can substantially affect the biomechanical properties of trabecular bone and its internal surface area is an important determinant of hormone responsiveness and of remodelling activity. Insight into 3D structure is possible by making use of the two dimensional (2D) information available from histological slides of bone samples, e.g. trans-iliac bone biopsies and
using stereology which only requires perimeter and area measurements to be made (Parfitt et al 1983).
1.4.2 Preparation o f bone samples
Bone is composed o f cells (osteoclasts, osteoblasts and osteocytes) and a matrix of collagen with inorganic mineral salts deposited within it. These are a crystalline complex of calcium and phosphate hydroxides called hydroxyapatite (Caio(P0 4)6(OH)2) (Stevens and Lowe 1992) with approximately 38 % o f it being calcium (Bancroft and Stevens 1990). Bone collagen does not become mineralised as soon it is deposited. Unmineralised collagen or osteoid tissue forms a border, often called a seam, on surfaces of newly formed bone. This is normally no more than 15 pm thick and covers only a small proportion of the surfaces (Bancroft and Stevens 1990). Many histological techniques are available for bone. Some such as paraffin embedding require décalcification to remove mineral and soften the tissue. However for the study of the bone mineral itself and its relationship with non-mineralised elements, sections are usually prepared by techniques that do not interfere with the mineral substance. These are referred to by histologists as ‘undecalcified methods’. Soft embedding media such as paraffin wax are inadequate to prevent the tissue crumbling as it is cut and acrylic resins are now the most widely used embedding media for undecalcified bone. The processing of bone samples for histomorphometry is a long process requiring dehydration of the specimen in graded alcohols and infiltration with increasing concentrations of resin over a period of weeks. The hardened resin blocks are then sectioned on microtomes (precision motorised cutting machines) to yield slices a few microns thick which are fixed on slides and stained.
Chapter One______________________________________________________________________Introduction and Review
1.4.3 Parameters o f bone structure
A slice o f tissue on a slide is a 3D object, but if cut thinly enough it approximates a true geometrical section, and when looked at through the microscope it produces a 2D image. This image consists of profiles which are the projections of structures in three dimensions on to a plane. Stereology is the study o f how these profiles in the 2D image are related to the 3D structure which was sampled (Parfitt et al 1983). The calculation o f 2D quantities such as area and perimeter from the primary data is obtained from quantitative light microscopy. Important histomorphometric parameters for evaluating bone microarchitecture include trabecular width, number density, separation and perimeter.
An alternative method is to study nodes on images where the trabeculae have been reduced to a single pixel thickness or ‘skeletonised’. Structural elements or struts are classified according to whether they start or end in a node, free end or cortical bone (Compston et al 1987). These parameters provide information on connectivity of the trabecular network and on the number of struts but results may be difficult to interpret intuitively. Trabecular perforations without much loss of trabecular mass would be expected to result in increased nodes and free ends, whereas more drastic loss of connectivity by removal o f whole rods and plates would result in reduction o f nodes and free ends (Recker 1993).
1.4.4 Dynamic variables
One great advantage o f bone histomorphometry is the ability to provide information on bone formation and resorption. Tetracycline has the property that it is laid down on all bone surfaces where active mineralisation occurs. By giving tetracycline to the individual in two sessions with an interval of 10 days prior to the biopsy procedure the extent of active mineralising surfaces can be determined. By measuring the distance between the two tetracycline lines the amount o f bone formed by the osteoblasts
during the labelling interval can be obtained. In normals the total trabecular surface is theoretically renewed every two to three years (Eriksen 1986).
1.4.5 Limitations
The use o f image analysis systems with automatic or semi-automatic methods for segmentation and analysis of video camera images o f bone slides has reduced the time required for measurement. However the time required for sample processing and the destructive nature of this processing are the main drawbacks o f this technique. There is a limit to how many bone biopsies can be taken from a subject and by the nature of the investigation they cannot be taken from the same place. Another limitation is that the technique is a 2D representation of a 3D structure. In spite o f these points, histomorphometry provides a means for the direct measurement o f bone and produces detailed information about bone microstructure.