Histomorphometric methods

Histomorphometry involves microscopic measurements o f histologic sections. Measurement of structurally visible or displayed objects or features is performed by

using eye-piece micrometers and eye-piece graticules or grids, with a specific geometry or pattern, covering the visible field of measurements. By point or linear intercept counting o f microscopic structures falling on a grid pattern, i.e. relative values o f area, perimeter or boundaries and the number of selected features may be obtained. The advantage is the opportunity to obtain qualitative and quantitative analyses o f a section. In recent years, advances in computerised image analysis systems have meant a progress in morphometrical techniques. Computer-based systems, with video cameras as scanning devices, generate images converted into digital values in array o f picture elements (pixels) and allow the assessment of area, intercept, perimeter, size and density values automatically or semi-automatically with suitable software. (Revell, 1983). However, the method can be limited by the ability to discriminate between structures, by section thickness and projection errors, by the method o f calibration. Counting is performed on two-dimensional data. Sample handling, fixation, staining and sectioning are factors that must be properly controlled.

The parameter most commonly used for assessing the bone-implant interface is the percentage o f the perimeter of an implant in contact with mineralised bone in longitudinal ground sections observed with light microscopy (Weinlaender et al., 1992; Cabrini et al., 1993; Lew et al., 1994; Hetherington et al., 1995; Nimb et al., 1995; Huré et al., 1996; Wennerberg et al., 1996a; Ivanoff et al., 1997). Such measurements still rely on the human observer to distinguish between what is and is not direct contact between implant and bone. In addition to measurement o f bone contact lengths, the area of bone formed within the threaded portions o f endosseous implants was routinely measured by the Gothenburg group led by Albrektsson (Johansson et al., 1990; Gottlander & Albrektsson, 1991; Wennerberg et al., 1996c). Members of the group have looked into technical factors which could affect the outcome of histomorphometrical quantifications of the bone-implant interface in ground sections. Section thickness and cutting directions were investigated (Johansson and Morberg, 1995a, 1995b). They concluded that sections over 30pm thick could result in overestimation of the bony contact. In their rabbit tibia model, 25% more contact lengths were found when the implants were cut in the longitudinal direction of the tibia than those cut in the transverse direction. This was thought to be due to the variation in the mode of loading of transcortical implants in the

rabbit femur model. Barzilay et al. (1996) compared the use of decalcified thin sections (4pm) and ground sections (30 - 150pm) and found no significant differences between the two histologic techniques in the bone:implant interface ratio which either technique provided.

The type o f implant material may also have an effect on the measurement procedure. Evans et al. (1996) have found that interpretation of the percentage of bone- implant contact was more difficult when evaluating HA-coated implants as compared to titanium implants since they sometimes experienced difficulty in distinguishing the HA- bone interface. Also to be considered is the site of implantation. Percentage figures of bone-implant contact are likely to be higher for implants or parts of implants residing in cortical rather than cancellous bone (Hipp & Brunski, 1987). Johansson et al. (1990) chose to measure bone-implant contact of threaded implants where they cross the cortical region of the tibia in their rabbit model for the reason that there was no cancellous bone in the marrow cavity where part of the implants were sited.

1.5,4.5 Tracer methods

Newly formed bone may be labelled with the aid of a tracer. The tracer may be detected in the light microscope due to fluorescing properties or with the aid of a radiation detector due to radioactivity.

• Fluorochrome labelling

Tetracyclines, alizarins, calceins, hematoporphyrins and xylenol orange are examples of non-radioactive, in vivo tracers of bone formation, where tetracyclines are the most frequently and widely used bone markers. They have common properties such as binding to calcium during mineralisation or demineralisation of bone and they fluoresce under UV-illumination. Tetracyclines bind to mineral in two ways, firstly, to the bone surface mineral front at the time of peak blood level in a reversible manner and secondly by trapping of new mineral deposited after the peak blood perfusion. The fluorochromes, usually tetracyclines, may be administered in sequences at regular time intervals yielding bands of labelled new bone or continuously during the the healing

period. Polyfluorochrome labelling may also be performed, by administering different tracers in specific schedules. Goldberg et al. (1995) used fluorochrome uptake to demonstrate the remodeling of bone on the surface of grit-blasted titanium implants in rabbit femurs. Haider et al. (1993) compared external and internal drill cooling methods for the preparation of holes to receive implants. By means o f polyfluorochrome sequential labeling, they found that patterns of new bone formation in cortical and cancellous bone differed according to the cooling method used.

• Radionuclide labelling

Radioisotope labelling has been used in qualitative and quantitative studies of bone healing. The detector systems generally used in the analysis o f bone repair are photographic emulsions. Organic matrix formation may be studied by tritiated amino acids, as ^H-glycine or ^H-proline in collagen synthesis analysis and ^^S-sulphate in proteoglycan or ground substance formation. Proliferative cell activity in fracture repair may be assessed by ^H-thymidine, labelling DNA synthesis.

An elegant study by Clokie & Warshawsky (1995) employed ^H-proline labelling to reveal the pattern of new bone formation during six weeks following the implantation of titanium implants in rat tibia. Radiolabelled new bone was deposited only on previously existing bone and extended towards the available space, leading to the suggestion that titanium does not influence the direction of bone growth in threaded implants but provides an inert template around which normal bone healing takes place.

1,5,4,6 Scanning Electron Microscopy

Scanning electron microscopy (SEM) is a useful tool for observing the topography o f implant surfaces either before or after implantation (Boyde, 1991). It is extensively used in secondary electron imaging mode for visualisation of the morphology of cells and extracellular matrix on implant materials. Boyde (1991) has warned against artefacts which are liable to occur at the junction between tissue and implants because of their differential shrinkages during specimen preparation. In fact, mechanical separation by fracturing is often necessary to expose the implant-tissue interface.

Back-scattered electron (BSE) images can help to distinguish bone with different mineral densities in addition to offering topographical contrast. When the surface o f the material is absolutely flat, or has been embedded, cut and polished flat, and viewed normally to the electron beam, the BSE image would be density-dependent. Nanci et al. (1994) have used this method to examine resin embedded sections o f the bone adjacent to screw-shaped titanium implants in rat tibia and to visualise the distribution o f silver- enhanced protein A-gold immunolabels for a series o f bone matrix proteins in such sections.