A computerised technique for the quantitative assessment of resorption cavities in trabecular bone

Bone, 11, 241-245 (1990)

8756-3282/90 $3 .00 + .00 Copyright © 1990 Pergamon Press plc

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A Computerised Technique for the Quantitative Assessment of Resorption Cavities in Trabecular Bone N. J . GARRAHAN, P . 1 . CROUCHER and J . E . COMPSTON Dept . of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN UK Address for correspondence and reprints : Dr . J. E . Compston, Dept . of Medicine, University of Cambridge Clinical School, Addenbrooke's

Hospital,

Hills

Road, Cambridge C92 2QQ, UK . report a new semi-automated method that enables a relatively rapid and more widely applicable quantitative assessment of several resorption cavity characteristics, including mean and maximum depth, length, and area .

Abstract A computerised technique is described for the quantitative assessment of resorption cavities in iliac trabecular bone . Using an /bas H image analyser, the original bone surface, eroded by bone resorbing cells, is reconstructed using a curve fitting technique that maintains a smooth continuity with the trabecular bone on either side of the cavity . Resorption depths are measured using an interactive elastic circle; all identified cavities are measured regardless of whether or not resorption is complete, and the measurements made include mean and maximum cavity depth, cavity length, area, and adjacent trabecular widths . Results in 13 normal subjects are presented. The technique is reproducible, simple to operate, relatively rapid, and can be applied to less sophisticated image analysis systems.

Methods Trans-iliac biopsies were obtained with a 6 or 8 mm internal diameter trephine one inch below and behind the anterior superior iliac spine . Biopsy specimens were fixed in 10% phosphate-buffered formalin and embedded in methylmethacrylate (BDH Chemicals Ltd ., Poole, Dorset) . Eight µm undecalcified sections were stained with 1% toluidine blue (pH 4 .2) . Resorption cavities were identified at a magnification of x 375 or, occasionally, X 750, under polarised light; all crenated surfaces with lamellae whose end points terminated at the edge of the trabecular bone surface were included (Vedi et al . 1984) . Between two and eight sections were examined from 13 biopsies, the mean number ± SD of resorption cavities identified in each biopsy being 24.3 ± 4 .2 . The biopsies were obtained from 13 normal subjects, 8 female and 5 male, aged 22 to 68 years, who formed part of a larger study of normal subjects reported in detail previously (Vedi et al . 1982) . An lbws II image analyser (Kontron, West Germany) was used, comprising an overall control unit (MOP Videoplan) together with a TV image capture and processor unit . The image of the resorption cavity is stored in the memory of the /bas 11 and its contents displayed on a TV monitor . Interactive graphics can be superimposed over the image using a screen cursor manually controlled from a digitiser tablet . The screen cursor is moved to the two end points of the resorption cavity and the X . Y coordinates of their positions entered into the image analyser. Two circles are automatically drawn on the screen, centred at each end point and having a diameter equal to the linear distance between the end points . The positions of the intersections between the circles and actual trabecular surface are entered using the cursor . A smoothly continuous curve (cubic spline) is calculated and drawn on the screen so that it passes through the four defined points . If the curve is not aligned accurately along the trabecular surface then the positions of maximum deviation can be added as additional points to refine the curve fitting procedure . It was found that good curve alignment could be obtained with up to a maximum of three points on each side of the resorption cavity even when the cavity was situated near the tip of a trabecula ; clearly,

Key Words : Histomorphometry-Taabecular bone-Resorption depth-Resorption cavity,

Introduction Bone remodelling occurs at discrete sites along the trabecular bone surface, resorption of a quantum of mineralised bone being followed by formation and subsequent mineralisation of osteoid within the cavity formed, so that under normal circumstances the amounts of bone resorbed and formed within each remodelling unit are quantitatively similar (Frost 1963) . If, within each unit, the amount of bone formed is less than that resorbed, irreversible bone loss results ; hence measurement of the amounts of bone resorbed and formed per remodelling unit provides important information about mechanisms of bone loss in osteoporosis, both at the cellular and structural levels . Completed bone packets (bone structural units) can be relatively easily identified under polarised light and measurement of their mean width, which represents the amount of bone formed per remodelling unit, is now a standard histomorphometric technique (Lips et al . 1978; Darby and Meunier 1981) . However, direct measurement of resorption depth has proved less easy, partly because of the greater difficulty involved in accurate identification of resorption cavities . One approach has been to count the number of eroded lamellae beneath the trabecular surface in resorption cavities containing preosteoblastic cells (Eriksen et at . 1984) . Such cells may, however, be difficult to identify and this technique has not been widely adopted . We 241

N . J . Garrahan et al . : Measurement of resorption depth

24 2

(a)

(b) Fig. 1 . Reconstruction of original bone surface using the computerised curve fitting technique on two resorption cavities . Good fitted curves are obtained both when the original trabecular outline is convex (a) or concave (b) . Magnification x 375 . resorption cavities that had resulted in trabecular penetration could not be identified and measured . The portion of the curve between the cavity end points is used as the baseline for measurements . Figure 1 shows examples of fitted curves at resorption cavities . Cavity depth and trabecular width measurement were carried out using an interactive elastic circle technique . This method involves anchoring a circle at the start point of a surface and interactively adjusting the centre and diameter of the circle until it is simultaneously tangential with the opposing surface . The linear distance between the tangential points was taken as the width or depth measurement . Maximum resorption depth and the mean depth of four approximately equidistant points along the

resorption cavity were measured together with the trabecular widths at the end points of the cavity (Fig. 2) . Studies of cumulative maximum and mean depths demonstrated steady values after measurement of 20 resorption cavities . All width measurements were multiplied by ar/4 to correct for obliquity of the plane of section . For assessment of the eroded surface (% total trabecular bone surface) and cavity count (per mm of trabecular bone surface or per mm2 trabecular bone), images produced on the TV screen were stored and segmented to produce a binary image . The total bone perimeter was then measured automatically, the above indices being calculated using data produced by the resorption cavity analysis . The following indices were obtained by mea-

N . J . Garrahan et al . : Measurement of resorption depth

243

Fig . 2 . Computerised measurement of resorption depth and adjacent trabecular widths . The maximum cavity depth (a), mean depth (b) (mean of four approximately equidistant points), and trabecular width at each end of the cavity (c) are measured using an interactive elastic circle . Magnification x 375,

surement or calculation : Cavity count/mm-no . cavities per mm trabecular bone surface Cavity count/mm'-no . cavities per mm 2 medullary area (trabecular bone + marrow) Maximum depth (µm)-maximum depth of cavity, measured interactively Mean depth (pan)-mean depth, derived from measurements made interactively at four equidistant points Reconstructed length (µm)-length of resorption cavity measured along computer-drawn line Original length (µm)-length of base of resorption cavity, measured interactively Cavity area (µm2 )-area of resorption cavity, measured automatically Eroded surface (%)-percentage of trabecular surface occupied by resorption cavities Trabecular width (µm)-trabecular width measured interactively at each end of the cavity The total time taken for comprehensive assessment of resorption cavity characteristics in one biopsy was 2 to 2 .5 hours .

tested on 29 clearly identified and photographed cavities, the same observer making measurements on two occasions six weeks apart . In this part of the study (reproducibility from photographs) the unit of observation was the cavity and the sample size N was 29 ; logarithmic transformation was used on all individual values because of non-normal distribution of the variables examined . For both parts of the reproducibility study, the mean of duplicate measurements (y) and its standard deviation (s), representing the distribution of the measurement between different determinations were calculated . The geometric mean was then calculated as 107, the fitted model predicting that values for 95% of the cavities lay in the range 107 ± 1 .965 . To assess reproducibility, paired differences (d) were calculated and the mean difference (d) and its standard deviation (Sd) then assessed . Significance of paired differences were examined using a paired t-test as follows : t = d /(Sd /-\/ N ) The standard deviation of a single reading about the true value attributable to measurement error, on a log scale (Sd*) was calculated as follows : Sd* = Sd/\/2

Reproducibility and Statistical Analysis

Reproducibility was tested in sections from six biopsies, duplicate measurements being made at an interval of six weeks by the same observer . In this part of the study (reproducibility of biopsy study) the unit of observation was the biopsy and the sample size N was 6 . Median values were used to express cavity variables within a biopsy and these medians were analysed using logarithmic transformation (log in) . Since individual cavities within biopsies were not labelled, the cavities measured on the two occasions might have varied in addition to any variations caused by measurement error . For this reason, reproducibility was also

The dimensionless measure of reproducibility generalising the coefficient of variation was then obtained as 100 k Sd*, where the factor k = 2 .3026 converts to a natural log scale .

Results The measured and derived resorption cavity characteristics in the 13 normal subjects are shown in Table I. Distribution of maximum cavity depth, mean depth, reconstructed and cement

N . J . Garrahan et al.! Measurement of resorption depth

2 44 Table 1 . Resorption cavity characteristics in 13 normal subjects .' Number of cavities counted Maximum depth (µm) Mean depth (µm) Reconstructed surface length (µm) Cement line length (µm) Cavity area (µm 2) Eroded surface (%) Cavity count/mm' Trabecular width (µm)

24 .4 e 4 .2 28 .9 (23 .4-39 .3) 15 .6 (13 .0-22 .0) 229 .5 (156-311 .7) 247 .7 (177 .6-332 .6) 4752 (2913-7322) 1 .35 ± 0 .39 0 .060 ± 0 .017 120 .0 (5 .2-548 .0)

*Results are expressed as mean ± SD or median (range) .

line length, and cavity area showed skewness to the left while the remaining indices showed a normal distribution . Reproducibility of the method (intra-observer variation) is shown in Table II . Duplicate measurements over an interval of six weeks did not reveal any systematic measurement error in either the biopsy or photographic study . The reproducibility of the cavity area measurement was relatively poor, but for other indices was reasonably good, and comparable to those reported for conventional histomorphometric variables . Overall, variation between biopsies was not greater than that between cavities ; although this might appear surprising at first sight, because identification of individual cavities must be a potential source of error, it can be attributed to the averaging process in the between biopsies study, each biopsy being characterised by the median of a large number of cavities .

Discussion The method we have described provides a relatively objective and rapid technique for the quantitative assessment of resorption cavities in trabecular bone . It can be applied to less sophisticated image analysis systems widely used in bone histomorphometry and enables a more comprehensive analysis of resorption cavity characteristics than has previously been attempted . As with other histomorphometric measurements of width, such as mean wall width and mean trabecular width, the method we describe is two-dimensional and extrapolation to three-dimensional structure is subject to some degree of error, which can be reduced to some extent by correcting for obliquity of the plane of section . Boyde et al . (1989) have recently described a method based on confocal scanning electron microscopy, in which a three-dimensional image of resorption cavities is obtained, from which measurements of depth and volume can be made . The reproduc-

ibility of the computerised technique reported here is acceptable and compares well with the method described by Eriksen et al . (1984) and also with that reported for conventional histomorphometric indices (Birkenhager-Frenkel et al . 1976, Compston et al . (1986) .

In the present study no attempt was made to identify different cell types, as described by Eriksen et al . (1984), nor to assess whether or not the resorption process had been completed . Thus all identifiable resorption cavities were included in the measurements ; the inclusion of cavities in which resorption had not yet been completed accounts in part for the lower mean depth values obtained compared with those reported by Eriksen et al . (1984) for cavities containing preosteoblastic cells, in which resorption was assumed to be complete . In his study, values for mean depth in these completed cavities were, as would be expected, similar to those reported for mean wall width, which represents the amount of bone formed within each bone remodelling unit . In contrast, our figures for mean resorption depth are considerably smaller than those we have reported for mean wall width in the same group of subjects (Vedi et al . 1983), because while mean wall width represents the amount of bone formed at completion of bone remodelling, resorption cavity depth was measured at all stages of the resorptive phase of remodelling . Another factor that may contribute to the lower values obtained for mean depth in the present study compared with those reported by Eriksen et al . (1984) is the exclusion by the latter authors of cavities from which recognisable cells (osteoclasts . mononuclear cells, and preosteoblasts) were absent . Such cavities amounted to approximately 25% of all those identified and represent a possible source of measurement bias that would lead to overestimation of mean values if smaller cavities were omitted . In Eriksen's study, 14% of cavities contained osteoclasts, 67% mononuclear cells, and 19% preosteoblastic cells with mean depths of 19, 49 .1, and 62 .6 p .m respectively, the relative frequency of these different stages of resorption being consistent with the calculated median function periods of the three phases . In contrast, in the present study only 5 .4% of cavities had a mean depth of 50 µm or more . Methodological differences in actual measurement technique are unlikely to account for these differences; the selection of resorption cavities for measurement, which has a large subjective element, particularly if the recognition of different cell types is included, appears a more likely explanation . The values for mean resorption depth recently reported in normal subjects by Palle et al . (1989) are closer to those obtained in the present study ; thus the mean corrected value in young males was 26 .0 µm and, in older males, 22.9 p.m . The method used in their study is not described in detail, but appears to have been based on counting the number of eroded lamellae beneath the trabecular bone surface in all identifiable resorption cavities .

Table II . Reproducibility of resorption cavity measurements . Between Biopsies Geometric Mean Number of cavities counted Maximum depth (µm) Mean depth (µm) Reconstructed surface length (µm) Cement line length (µm) Cavity area (µm) 2 Eroded Surface (%) Cavity count/mm 2 Cavity count/mm Trabecular width (µm)

23 .4 24 .0 14.0 206.0 231 .0 3017 1 .44 0 .248 0 .064 112 .1

(N =

6)

Between Cavities

(N =

29)

95% Range

CV%

Geometric Mean

95% Range

CV%

14 .7-38 .3 18 .7-30 .7 10 .0-19 .4 145-292 166-320 1753-5192 0.79-2 .64 0.121-0 .510 0.037-0 .109 82 .6-244

6 .8 18 .4 20 .8 13 .4 20 .0 34 .6 25 .6 13 .9 14.8 8 .7

35 .2 21 .2 283 .0 231 .0 4794

12 .9-95 .6 7 .6-59 .3 104-768 76.5-701 850-7040 -

13 .4 12 .9 6 .5 31 .5 44 .5

136 .4

66 .3-280.5

7 .5

N . J . Garrahan et al . : Measurement of resorption depth

Using the computerised technique described here we were able to assess quantitatively a number of resorption cavity characteristics not previously reported, for example resorption cavity length and area . In particular, the maximum cavity depth is an important determinant of trabecular penetration which, up until now, has not been assessed . When measurements on individual cavities were examined a surprising range of values was revealed, maximum depths up to 96 .0 p.m being found in some cavities in bone from normal subjects . These values are considerably in excess of maximum mean cavity depth values (59-70 µm), indicating that median values for mean depth obtained from a number of values in one biopsy may mask important intra-individual variations in cavity dimensions within a single biopsy, and may also underestimate the likelihood of trabecular penetration . Although considerable advances in bone histomorphometric techniques have taken place over recent years, the assessment of resorption has remained unsatisfactory . Measurement of resorption depth, which provides valuable information about mechanisms of bone loss in osteoporosis and its accompanying structural alterations, has only recently been attempted (Eriksen et al . 1984) . However, this technique is difficult and time consuming, and has not become a standard histomorphometric procedure . Resorption depth has also been assessed indirectly by calculation of mean interstitial thickness from values obtained for mean wall thickness and trabecular width (Courpron et al . 1980) ; however, the relationship between mean interstitial thickness and resorption depth is not a simple inverse one (Croucher et al . 1989) . The computerised technique described in this paper is widely applicable to image analysis systems used by bone histomorphometrists, and should enable assessment of the contribution of increased resorption depth to bone loss in different types of osteoporosis .

Acknowledgments : We are grateful to the Welsh Office for financial

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variation and intrinsic error of measurement for some parameters ; within single bone biopsies . Bone Histomarphomeny. Second International Workshop . Meunier, P . J., ed . Toulouse, France : Societe de Is Nouvelle Imprimerie Foumie ; 1976:63-67 . Boyde, A. ; Jones, S . J . ; Dillon, C . An automatic method for 3-D characterization of osteoclastic resorption lacunae, Bone 10:150 ; 1989 . Compson, J. E . ; Vedi, S . ; Stellar, A. 1 . Inter-observer and intro-observer variation in bone histomorphometry . Calcif. Tissue Inc. 38 :67-70 : 1986 . Courpron, P. ; Lepine, P . ; Arlor, M . : Lips, P . ; Meunier, P . 1 . Mechanisms underlying the reduction with age of the mean wall thickness of trabecular basic structure unit (BSU) in human iliac bone . Bone Histomorphomerry . Third International Workshop. lee, W. S . S . ; Purist, A . M ., eds . Paris : Armour Montagu : 1980:323-329 . Croucher, P . 1 . : Mellish, R . W. 6 . ; Vedi, S . ; Garrulous, N. J . ; Compvton, J . E . The relationship between resorption depth and mean interstitial bone thickness (MIBT) : age-related changes in man . Calcij. Tissue Ins . 45:15-19 ; 1989. Darby, A . J. ; Meunier, P . J. Mean wall thickness and formation periods of trabecular bone packets in idiopathic osteoporosis . Calcif. Tissue Ins . 33 : 199-204; 1981 . Erdcsen, E. F . ; Melon F. ; Mosekilde, L . Reconstruction of the reaorptive site in iliac trabecular bone : a kinetic model for bone resorption in 20 normal individuals . Merab. Bane Dis. & Rel. Rec . 5 :235-242; 1984 . Frost . H . M . Bane Remodelling Dynamics . Springfield, Illinois : Charles C Thomas; 1963 . Lips. P . ; Courpron, P. ; Measlier, P. J . Mean wall thickness of trabecular bone packets in the human iliac crest : changes with age. Calcif. Tissue Res . 26 :13-17; 1978 . Palle, S . ; Chappard, D . ; Vico, L . ; Riffat, G . ; Alexandre, C. Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects : a hismenzymologic and histomorphometric study . J. Bone Min . Res . 4 :501-506 ; 1989 . Vedi, S . ; Compston, J . E . ; Webb, A . ; Tighe, J . R . Histomorphometric analysis of bone biopsies from the iliac crest of normal British subjects . Merab . Bone Dis . & Rel. Res . 4 :231-236 : 1982 . Vedi, S . ; Compston, 1 . E. ; Webb, A . ; Tighe. J . R . Histomorphometric analysis of dynamic parameters of trabecular bone formation in the iliac crest of normal British subjects . Merab. Bone Dis . & Rel . Res . 5 :69-74; 1983 . Vedi, S . ; Tighe, J . R . ; Compston, J . E . Measurement of total resorption surface in iliac crest trabecular bone in man . Merab. Bone Dis . & Rel . Res . 5:275-280 ; 1984 .

support .

References Received: October 6, 1989

Birkenhager-Frenkel, D . H . ; Schantz, P . I . M. ; Breuls, P . N . W . M . ; Lockefeer, 1 . H. M. ; Heat, vd . R. O . Biological variation as compared to inter-observer

Revised: March 13, 1990

Accepted: March 22, 1990