Methods for the imagine of bone

Bone, 13, pp. 95-106 (1992) Printed in the USA. All rights reserved.

Copyright

8156-3282192 $5.00 + .OO 0 1992 Pergamon Press plc

IMAGING OF CARTILAGE AND BONE MEETING, JUNE 27, 1991 Venue: Institute

Local Organizers:

of Orthopaedics S. Downes,

(UCMSN),

R. S. Archer,

Royal National

M. V. Kayser,

C. J. Clifford,

Orthopaedic

Hospital,

C. A. Scotchford.

Stanmore,

Middx.,

UK

J.-P. Cassella

video-captured images to produce three-dimensional reconstructions of objects in bone like osteoclast resorption pits. It is also possible to visualise and record fine details of structure in living cells in real time using confocal microscopy linked to videotape image storing facilities. Transmission electron microscopy enables the recognition of cell types and cytoplasmic details, examination of bone collagen, and its mineralisation. At high power ( X 1OOK) bone mineral crystals are recognisable, and at even higher magnification (X 2M), crystal lattices can be visualised. Negative staining (with sodium silicotungstate) enables recognition of membrane bound structures, most notably matrix vesicles. Scanning elecron microscopy (SEM) at low power offers little more than dissecting light microscopy but, as the magnification is increased, collagen fibre patterns can be recognised and focal sites of calcification on individual collagen fibres are demonstrable. SEM may also be used to demonstrate the tissue reaction of bone to implanted materials by examining the surface of plastic-embedded tissue blocks. Osteoclasts cultured on slices of bone can be demonstrated by SEM to produce resorption pits with a characteristic appearance. Application of SEM to the study of paleopathological material makes it possible to recognise and differentiate vascular channels, osteoclastic resorption sites, and erosive damage caused by soil. Further techniques include freeze fracture of the sample, followed by SEM, for example, to demonstrate budding of the chondrocyte cytoplasmic membrane to form matrix vesicles. The base scattered image in the SEM comes from the first few microns of the surface of a polished plastic-embedded tissue block. This technique can produce an image equivalent to a high-resolution microradiograph of bone. Such a back scattered SEM image can also be processed by computer method to provide a pseudo-coloured chart showing relative bone density.

Methods for the imagine of bone. P.A. Revel1 Department of Histopathology, London Hospital Medical College, London El 2ALI Techniques for the imaging of bone are available at all levels, from simple low-power microscopy and the naked eye appearances through to sophisticated high-technology methods. Although the use of expensive equipment does much to enhance our understanding of the structure of bone, the amount of information available by simple visualisation is well illustrated, for example, by examining samples of bone remodelled around an implanted screw, or the surface of a titanium alloy implant cemented into bone with acrylic. For light microscopy, decalcified or undecalcified sections are chosen according to the particular needs of the problem being examined. Thin plastic embedded sections of undecalcified bone enable fine details of cells to be visualised, and special staining techniques such as Goldner’s trichrome, von Kossa, toluidine blue, or thionin to be applied. Such techniques are vital for histomorphometric analysis of bone, enabling the easy recognition of features for capture by a video system and computerised image processing. Special staining methods are also available for the demonstration of aluminum or iron in bone. This thin plastic embedded sectioning technique is also particularly suitable for the study of growth plates in the chondrodysplasias. Enzyme histochemistry can be applied to bone to demonstrate tartrate-resistant acid phosphatase in osteoclasts and alkaline phosphatase in osteoblasts. Polarisation microscopy is useful for the recognition of crystals (for example, calcium oxalate) and the demonstration of the patterns of collagen fibre orientation in bone. Metal implants may be sectioned in bone, with or without various surface treatments (e.g., hydroxyapatite coatings) by using plastic embedding and specialised cutting devices or milling machines. Another technique is ultraviolet light microscopy for the localisation of fluorescent tetracycline label in bone at sites of active calcification. Double labelling with tetracycline enables the estimation of bone appositional rates. Although there are some problems relating to the use of immuno-histochemical techniques in bone, monoclonal antibody (MAb) labelling can be applied to soft tissue samples from implant interfaces and a number of MAbs also are reactive in decalcified tissue sections. A specialised, recently developed area of microscopy at the light level is confocal microscopy, which is either of the tandem scanning or laser scanning type. These techniques allow line focussing and clear visualisation through a depth of focus, either with conventional light or a laser beam. Confocal microscopy can be combined with computerised processing of

Acknowledgments:It is a pleasure to acknowledge the help given to me by Professors Ali, Anderson, and Boyde, and Dr. Tanner, all of whom generously enabled me to use examples of their work to illustrate this presentation.

A variety of techniques for visualising bone resorption by human osteoclasts. J.A.S. Pringle Departmewof Morbid Anatomy, Stanmore, Mid& HA7 4LP

Instituteof Orthopaedics(UCMSM),

It is now almost ten years since the first in vitro resorption of bone by human osteoclasts was achieved using osteoclast giant 95