JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 9. 1991 Mary Ann Liebert, Inc., Publishers
Relationship Between the Number of Resorbing Cells and the Amount Resorbed in Metabolic Bone Disorders MARTINE COHEN-SOLAL, CAROLINE MORIEUX, and MARIE-CHRISTINE DE VERNEJOUL
ABSTRACT The relationship between bone-resorbing cells, assessed by the presence of tartrate-resistant acid phosphatases (TRAP) and morphologic indices of bone resorption, was determined in 29 osteoporotic patients (14 postmenopausal females and 15 males) and 15 dialyzed patients. The number of TRAP-positive cells per unit of cancellous bone area (N.Oc/B.Ar) was higher in dialyzed patients than in those with osteoporosis (16.8 k 15.3 versus 4.95 + 2.86, p < 0.05). The amount of bone resorbed at the basic multicellular unit level was estimated by calculating eroded area containing TRAP cells per bone area (E.Ar+/BA). This novel parameter was similar in dialyzed and in osteoporotic patients (41,700 k 28,400 versus 32,300 k 24,600). In contrast, trabecular spacing (Tb.Sp) was identical in both metabolic bone diseases. Trabecular width (169 + 38 versus 127 k 32 pm, p < 0.05) and bone area were higher in dialyzed than in osteoporotic patients. N.Oc/B.Ar was significantly related to E.Ar+/BA in dialyzed ( r = 0.76, p < 0.05) but not in osteoporotic patients. Tb.Sp was significantly correlated to N.Oc/B.Ar and to the number of TRAP-positive cell nuclei per B.Ar ( r = 0.44, p < 0.05) in osteoporotic but not in dialyzed patients. This last result shows that in overt osteoporosis with thin trabeculae, trabecular spacing is related to the number of resorbing cells. In contrast, the spacing of thick trabeculae in dialysis osteodystrophy is not dependent on the number of osteoclasts.
INTRODUCTION
B
is the end result of the balance between osteoblast and osteoclast activity at the tissue level. The relationship between bone cell number and other morphologic indices is therefore an important point in understanding the mechanisms of bone loss. During aging and in osteoporosis, cancellous indices related to bone formation are diminished: trabecular and wall thickness decrease with age and are further decreased in osteoporotic patients The imcompared to age-matched control paired bone formation in osteoporosis is associated with changes in cell number: we observed a decreased osteoblast that was associated with a decreased in vitro proliferating capacity of osteoblasts isolated from bone biopsies. 1 5 ) The increase in trabecular spacing, a structural change occurring during aging and osteoporosis, is believed to be a major determinant of bone and may decrease O N E MASS
bone strength. It has been related to osteoclastic trabecular perforation during the years following the menopause. 1 2 ) To test the hypothesis that the osteoclast activity plays a role in our population with overt osteoporosis, we examined the relationship between the resorbing cells identified by the presence of cytoplasmic tartrate-resistant acid phosphatase (TRAP) and morphologic indices of cancellous bone. We studied three different metabolic bone states: ( 1 ) renal osteodystrophy, a condition not associated with bone loss"); (2) osteoporosis; and (3) normal subjects. Our data show that, in the osteoporotic patients only, trabecular spacing is related to the number of resorbing cells and resorbing cell nuclei.
PATIENTS AND METHODS
Patients and control subjects We studied three groups of patients. The first group consisted of 29 osteoporotic patients who had a bone bi-
INSERM Unite 18, Centre Viggo Petersen, Hapita1 Lariboisiere, Paris, France. 915
COHEN-SOLAL ET AL.
916
opsy for evaluation of bone status. All presented one or more vertebral compression fractures on spinal x-ray. They were divided into two groups: 14 women (mean age 68.7 k 8 years) who had undergone menopause 10 years earlier and 15 men (mean age 56 f 12) with idiopathic osteoporosis but without endocrine disease or corticosteroid therapy. However, 6 of them gave a history of chronic alcoholism ( > 80 g/day) or tobacco ( > 20 packs a year) intoxication. The second group consisted of 15 dialyzed patients (mean age 60 k 8 years, mean duration of dialysis 114 f 77 months) for whom the bone biopsy was performed to assess a possible secondary hyperparathyroidism. A total of 21 healthy young men (mean age 32.8 + 4.7), used as controls (third group), were the subjects previously reported in the study of Palle et a1.I') The stained sections were kindly provided by Dr. Alexandre (Saint-Etienne, France).
Methods The transiliac bone biopsies were embedded in methyl methacrylate at low temperature with slight modifications of a method previously d e ~ c r i b e d . ' ~Sections ' were cut with a heavy microtome (type Jung K). The sections were stained using naphthol ASTR phosphate as a substrate for tartrate-resistant acid phosphatases1Io1and counterstained with toluidine blue, pH 2.4. All the histomorphometric measurements were performed on two or three distant sections using an objective eyepiece Zeiss Intergratte plate I1 and a semiautomatic image analyzer (ASM, Leitz, Germany). The measurements were performed on total cancellous bone, and results are expressed in the second dimension according to ASBMR standardized nomenclature. ( I L 1 The bone microstructural indices were calculated from the perimeter and area measurements according to Parfitt et al."': Bone area B.Ar/T.Ar To
=
Trabecular width Tb.Wi, Fm B.Ar/B.Pm
B.Ar/T.Ar x 100 =
2000/1.199
Trabecular number Tb.Nb/mm' B. Pm/T. Ar Trabecular spacing Tb.Sp, pm (T.Ar - B.Ar)/B.Pm
=
=
X
1.199/2 x
rotic and dialyzed patients; the count was not possible in the normal subjects because of a slight difference in the technical procedure. Oc.Pm/B.Pm: osteoclastic perimeter expressed as a percentage of total cancellous perimeter. E.Pm/B.Pm: eroded perimeter expressed as a percentage of total cancellous perimeter.
To assess the amount of bone resorbed, we performed several measurements on every eroded cavity (E) containing TRAP cells (+) or without TRAP cells (-). The cavities containing osteoid were excluded. For each cavity were measured The length of the previous quiescent surface by eye reconstruction following the shape of the trabeculae on either side (E.length- or E.length+). The mean depth (E.De- or E.De+) by measuring the orthogonal distances between the reconstructed length and the base of the cavity at equidistant points indicated by the line of the eyepiece graticule (Fig. 1). The eroded area (E.Ar- or E.Ar+) was calculated indirectly by multiplying the length of the reconstructed line and the mean depth: E.Ar-
=
E.length- x E.De-
E.Ar+
=
E.length+ x E.De'
The eroded area was also expressed as an index using the bone area as referent: E.Ar-/B.Ar
=
E.Ar-/(B.Ar/T.Ar)
E.Ar'/B.Ar
=
E.Ar+/(B.Ar/T.Ar)
Reproducibility and statistical analysis The intraobserver reproducibility of our method was assessed by remeasuring six bone biopsies at an interval of 1 month by the same observer. The same biopsies were measured by another observer trained by the first for interobserver reproducibility. The coefficients of variation were calculated according to Compston et aL1''' The results are expressed as means and standard deviations. The unpaired Student's f-test was used to test the significance of differences between groups. Correlations were assessed using linear regression analysis.
2000/1.199 x
The following measurements were performed at a x 160 magnification:
N.oc/T.Ar and N.oc/B.Ar: number O f osteoclasts per m m 2 tissue area and per m m 2 bone calculated by counting only TRAP-positive cells in cancellous area and using the tissue and bone area as referents. N.Oc.Nc/T.Ar and N.Oc.Nc/B.Ar: number of osteoclast nuclei per mm2 tissue area and per mm' bone area, calculated by counting the number of osteoclast nuclei in cancellous area and using the tissue and bone area as referents. Those variables were counted only for the osteopo-
RESULTS The results of the intraobserver reproducibility show a coefficient of variation of 9.68% for E.length-, 10.9% for E.length+, 12% for E.De+, and 13% for E.De-. For the interobserver reproducibility, the coefficient of variation was 14% for E.length-, 14% for E.length+, 15% for E.De+, and 10% for E.De. The microstructural indices (Table 1) showed that there were no differences in any variables between osteoporotic males and females; the data were therefore pooled. As expected, all the microstructural indices were lower in the
ASSESSMENT OF BONE RESORPTION
917
osteoporotic group than in the controls except trabecular spacing, which was increased. Compared to controls, patients with renal osteodystrophy had decreased trabecular number and increased trabecular spacing. Bone area, trabecular width, and trabecular number were higher in dialyzed patients than in osteoporotics, whereas trabecular spacing was not different. The resorption indices are reported in Table 2. The number of osteoclasts and osteoclast nuclei number were significantly higher in dialyzed compared to osteoporotic patients, whether expressed per tissue or bone area. Reflecting the heterogeneity in the renal osteodystrophy group, the range of osteoclast number values is wide in dialyzed patients: some patients had values as low as those in osteoporotics, whereas others had fivefold higher values. In the osteoporotic group, the slightly higher osteoclast number value in females compared to males was not significantly different. When osteoclast and osteoclast nuclei counts were expressed using bone area as referent, the women had higher osteoclast indices than the men. In both diseases, the Pumber of osteoclasts was increased compared to controls. E.De- compared to E.De' or E.Ar- compared to E.Ar+ was never significantly different in any of the three groups.
In the dialyzed patients, the erosion depth with osteoclasts was higher so that the absolute value of E.Ar+ is increased compared to that in osteoporotics and controls. When expressed with B.Ar as referent, however, there is no difference because of a lower bone area in osteoporotics. The eroded area with osteoclasts (E.Ar+/B.Ar) was significantly increased in osteoporotic patients compared to controls, also due to a smaller bone area, whereas the eroded and osteoclast perimeters of osteoporotics and controls were similar. To correlate cell or nuclei number and the other variables, only osteoclast number using bone area as referent was considered. In patients with renal osteodystrophy, the eroded area with osteoclasts was correlated with the osteoclast nuclei number ( r = 0.69, p < 0.01) and osteoclast number ( r = 0.76, p < 0.01; Fig. 2). However, the E.Ar-/ B.Ar and osteoclast indices were not correlated with trabecular width, number, or spacing. In contrast, in the control and osteoporotic groups, we could not find a relationship between eroded area with osteoclasts and the osteoclast indices. However, in the osteoporotics only, there was a positive correlation between Oc.Nb/B.Ar and Oc.Nc.Nb/B.Ar with the trabecular spacing ( r = 0.37, p < 0.05; r = 0.44, p = 0.01; Fig. 3).
DISCUSSION
FIG. 1. M~~~~~~~~~~ of eroded area. ~h~ previous quiescent surface was reconstructed following the curvature of the trabeculae. The depth was measured as the orthogonal distance between this line and the base of the cavity.
Renal osteodystrophy and osteoporosis are two markedly different disorders. In dialyzed patients parathyroid hormone is the main factor regulating bone resorption.' 1 3 ) In contrast, this calcitropic hormone is not the main factor regulating osteoclast number or activity in osteoporosis. The relationship between resorbing cells and the amount of bone resorbed in both circumstances could therefore lead to different data. Osteoclasts can better be determined by histochemical identification of TRAP-positive cells. Indeed, multinuclearity is an uncertain criterion, since it has been shown that most osteoclasts have no nuclei visible on the section or contain only one nuclei.f14)In this study, the
INDICES TABLE1. STRUCTURAL Osteoporotic patients Index B.Ar/T.Ar, % T b Width, pm Tb.Nb, mm' Tb.Sp, pm
Controls (n = 21) 28.6 159 1.66 449
f f f f
6 36.9 0.22 83
Females (n = 14)
Males (n = 15)
Combined (n = 29)
Dialyzed patienls (n = 15)
13.5 + 4.6 122 5 36 1.13 + 0.3 814 + 210
17.2 f 5.3 131 + 27 1.31 k 0.3 697 f 243
15.3 k 5.2a 127 k 32a 1.22 + 0.35a 757 f 230a
22.7 k 12b 169 f 38b.C 1.35 0.56b 660 k 250d ~~
a C ~ n t rversus ~ l ~ combined osteoporotic patients, p < 0.01. Wombined osteoporotic versus dialyzed patients, p < 0.05. 'Combined osteoporotic versus dialyzed patients, p < 0.01. dControls versus dialyzed patients, p < 0.01.
COHEN-SOLAL ET AL.
918 TABLE2. RESULTSOF
THE
RESORPTION INDICES
Osteoporot ic patients Controls (n = 21) N.Oc/T.Ar, mm-’ N. NcOc/T.Ar, mm-’ N.Oc/B.Ar, mm-* N.OcNc/B.Ar, mm-’ Oc.B.Pm/B.Pm, Vo E.B.Pm/B.Pm, Vo
0.79 f 0.37
E.De-, pm E.De+, pm E.length-, pm E.length+, pm E.Ar-, pm’ E.Ar+, pm’ E.Ar-/B.Ar, per lo00 E.Ar’/B.Ar, per lo00
23.6 f 22.5 f 223 f 247 f 5300 f 5600 f 1.93 f 2.01 f
-
2.91 f 1.56 -
1.69 f 0.73 3.77 f 1.46 6.9 4.1 55 44 2550 1670 1.14 6.54
Females (n = 14)
0.77 0.95 6.08 7.81 1.5 4.42
Males (n = IS)
Combined (n = 29)
f f f f f f
0.36 0.45 3.2 3.78 0.9 1.15
0.58 0.67 3.89 4.46 1.1 3.22
f f f f f f
0.27 0.38 2.1~ 3.le 0.5~ 0.93e
0.67 0.81 4.95 6.08 1.29 3.87
20.7 f 19.6 f 177 f 208 f 3740 f 4490 f 2.83 f 3.59 k
3.78 5.9 62 95 1620 3240
20.6 20.7 189 217 3910 4570 2.55 2.85
f f f f f f f
2.85 3.5 78 89 1730 2120 1.5 1.43f
20.7 f 3.31f 20.1 f 4.87 183 f 69f 212 f 91 3820 f 1650 4530 f 2710 2.69 f 1.27f 3.23 k 2.46f
104
3.15
k
f f f f f f
0.33 0.43 2.86d 3.79 0.79 1.52
Dialyzed patients (n = 15)
3.58 f 5.85 f 16.5 f 25.3 f 3 f 6.74 f
3.53a 6.74b 14.9a 24.6b 2.08b 2.3P
30 f 16.8~ 46.1 f 22.7a 157 f 7 9 182 57h 5110 f 2910 8430 f 5240i 2.94 f 1.09j 4.17 f 2.831
*
dDialyzed patients versus controls and combined osteoporotic versus dialyzed patients, p < 0.01. Wombined osteoporotic versus dialyzed patients, p < 0.01. LFemale versus male osteoporotic patients, p < 0.05. C o m b i n e d osteoporotic patients versus controls, p < 0.01. ‘Female versus male osteoporotic patients, p < 0.01. ‘Combined osteoporotic patients versus controls, p < 0.05. wDialyzed patients versus controls, p < 0.05, and combined osteoporotic versus dialyzed patients, p < 0.01 hDialyzed patients versus controls, p < 0.01. ]Dialyzed patients versus controls and combined osteoporotic versus dialyzed patients, p < 0.05. ]Dialyzed patients versus controls, p < 0.05.
amount of bone resorbed at the basic multicellular unit (BMU) level as evaluated by an original method taking into account not only the depth of the resorption cavity(8.1S) but also its length. Because the osteoclast is a highly motile the length of the lacunae also reflects the activity of the cell. Moreover, the eroded and osteoclast surfaces seem to be insufficient variables to assess the resorption: in the present study, for example, the E.Ar+ or E.Ar‘/B.Ar was higher in osteoporotics, despite the absence of difference in osteoclast or eroded perimeters. Such an attempt to measure resorption cavity has recently also been made by Garrahan et al.[17’The method we describe is very similar and indeed values observed concerning mean depth, length, and absolute area of the resorption cavities in normal subjects are very close in both studies. The slight difference may be due to automatic direct measurement[”) compared to calculated index in our study. However, reproducibility was similar in both methods. We separated values obtained in cavities either with or without TRAP-positive cells. We expected values of the mean depth and area of cavities devoid of TRAP cells to be higher because the resorption process is supposed to be achieved in these “reversal” This was not the case, neither in controls nor in any metabolic bone diseases. This might be because lacunae without TRAP cells may contain TRAP cells in another section plane. Another
possible explanation for this observation is that osteoclastic resorption may not be a continuous phenomenon during the resorption phase of the cycle: there would therefore be an “on-and-off’ phenomenon, as for formation.[18’ This would indicate that all the lacunae devoid of osteoclasts are not in the reversal phase of the remodeling cycle. In patients with renal osteodystrophy, there was a clear relationship between osteoclast number and eroded area with osteoclasts. This result suggests that, at the basic multicellular unit level, osteoclast activity is dependent on osteoclast number. Eroded area without osteoclasts was not correlated to osteoclast number. This significant relationship between osteoclast number and eroded area was not observed in osteoporosis. Taking into account osteoclast nuclei instead of osteoclast number did not give a significant relationship, either. This negative result may be due to variable osteoclastic activity among patients or to regulation by two different factors of osteoclast number and activity in osteoporosis. Structural indices of bone architecture reflect bone cell activity at the tissue level. They are useful indices of threedimensional microstructures and have shed new light on the mechanism of bone loss. In our study, bone area and trabecular width were higher in dialyzed patients whereas trabecular spacing was identical in both groups. Trabecular thickness has been shown to reflect bone formation and
919
ASSESSMENT OF BONE RESORPTION
E.Ar +I B.Ar
10
20
30
40
50
60
70
N.Od B.Ar (/ mm2)
FIG. 2. Relationship between osteoclast numberlbone area (N.Oc/B.Ar) and eroded area with osteoclast/bone area (E.Ar'/B.Ar) in 15 dialyzed patients: r = 0.76, p < 0.01).
4ooo1
':
**lo
,
2000
0
10
,
higher in renal osteodystrophy than in osteoporosis, but formation is also higher. This is due to the dual effect of parathyroid hormone in both formation and resorption. This suggests that the balance between resorption and formation is preserved in secondary hyperparathyroidism, as well as in primary hyperparathyroidism.l") Thick trabeculae cannot be perforated even in the presence of a large number of osteoclasts; indeed, in such circumstances trabecular spacing does not reflect the number of resorptive cell at the tissue level. During aging and in osteoporosis, Parfitt et al.'') have shown that bone loss is primarily caused by the complete loss of trabecular plates, which increase separation between remainders. In osteoporosis, mainly in female patients, trabecular spacing was further increased compared to that in age-matched controls.I2) I t has been proposed that trabecular separation could be explained by intense resorptive activity at the time of menopause." ' ) Because of the lack of an age- and sex-matched control group for the osteoporotic patients in this study, we cannot conclude whether it is related to increased erosion depth or increased resorption sites. Our data are in agreement with those of other studies showing thin trabeculae in osteoporosis'' '01 related to decreased bone formation. The close relationship observed between osteoclast number and trabecular separation suggests that trabecular separation by increased osteoclast number is not a phenomenon limited to the years following the menopause but a process still occurring in older female and male osteoporotic patients. The implication for osteoporosis treatment of such a structural consequence of increased osteoclast number might improve therapeutic efficacy in osteoporosis as increased trabecular spacing has been shown to be an important risk factor for fractures."" ' I )
ACKNOWLEDGMENTS
20
N.Oc.Nc/ B.Ar (/ mm2)
FIG. 3. Relationship between osteoclast nuclei number/ bone area (N.Oc.Nb/B.Ar) and trabecular spacing in 29 osteooorotic oatients: r = 0.44. D < 0.05).
The authors are fully grateful to Dr. Parfitt for reviewing the manuscript and to Drs. Alexandre and Chappard for providing the slides.
REFERENCES is related in normal subjects to wall thickness.IJ) Trabecular spacing was comparable in dialyzed and osteoporotic patients; however, osteoclast number was higher in dialyzed patients. It is not likely that this result is due to lower osteoclast resorption activity in dialyzed rather than in osteoporotic patients since the absolute value of eroded area with osteoclasts was higher in dialyzed patients than in osteoporosis. However, because the N .Oc/B.Ar is fivefold higher, we also expected to find E.Ar'/BA fivefold higher; it cannot be excluded that the osteoclast activity is slightly lower in renal osteodystrophy. An identical value of trabecular spacing in the face of a higher osteoclast number can be explained by a greater trabecular width in renal osteodystrophy. Moreover, osteoclastic resorption is
I . Lips P , Courpron P , Meunier P J 1978 Mean wall thickness of trabecular bone packets in the human iliac crest: Changes with age. Calcif Tissue Res 26:13-17. 2. Parfitt C , Mathews C H E , Villanueva AR, Kleerekoper M , Frame B, Rao DS 1983 Relationships between surface, volume, and thickness of iliac trabecular bone i n aging and in osteoporosis. J Clin Invest 73:1396- 1409. 3. Aaron J E , Makins NB. Sagreiya K 1987 The microanatomy of trabecular bone loss in normal aging men and women. Clin Orthop 215:260-271. 4. Garcia-Carrasco M, dc Vernejoul MC, Sterkers Y , Morieux C , Kuntz D, Miravet L 1989 Decreased bone formation in osteoporotic patients compared to age matched control. Calcif Tissue Int 44:173-175. 5. Marie P J , Sabbagh A, de Vernejoul MC, Lomri A 1989 Osteoblastic cells isolated from posf-menopausal osteopo-
COHEN-SOLAL ET AL.
920
6.
7.
8.
9.
10.
11.
12.
13.
14.
rotic women: Relationships between osteocalcin and DNA synthesis in vitro and histomorphometric indices of bone formation. J Clin Endocrinol Metab 69:272-280. Weinstein RS, Huton MS 1987 Decreased trabecular width and increased trabecular spacing contribute to bone loss with aging. Bone 8:137-142. de Vernejoul MC, Marchais S, London G, Bielakoff J , Morieux C , Miravet L 1985 Increased in stainable bone aluminum after subtotal parathyroidectomy in dialysed patients. Kidney Int 27:785-791. Palle S, Chappard D, Vico L, Riffat G, Alexandre C 1989 Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects: A histoenzymologic and histomorphometric study. J Bone Miner Res 4:501-505. Chappard D, Palle S, Alexandre C , Vico L, Riffat G 1987 Bone embedding in pure methyl methacrylate at low temperature preserves enzyme activities. Acta Histochem (Jena) 81: 183- 190. Baron R, Vignery A, Neff L, Silverglate A, Santa Maria A 1983 Processing of undecalcified bone specimens for bone histomorphometry. In Recker RR (ed.) Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, FL, pp. 13-35. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H. Meunier P , Ott SM, Recker RR 1987 Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 2(6):595-610. Compston JE, Vedi S, Stellon AJ 1986 Inter-observer and extraobserver variation in bone histomorphometry. Calcif Tissue Int 38:67-70. de Vernejoul MC, Kuntz D, Miravet L, Gueris J , Ryckewaert A 1981 Bone histomorphometry in hemodialysed patients. Metab Bone Dis 3:175-169. Baron R, Neff L, Tran Van P , Nefussi JR, Vignery A 1986 Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am J Pathol 122:363-378.
15. Eriksen EF, Mosekilde L, Melsen F 1985 Trabecular bone resorption depth decreases with age: Differences between normal males and females. Bone 6:141-146. 16. Kanehisa J , Heersche JNM 1988 Osteoclastic bone resorption: In vitro analysis of the rate of resorption and migration of individual osteoclasts. Bone 9:73-79. 17. Garrahan NJ, Croucher PI, Compston J E 1990 A computerised technique for the quantitative assessment of resorption cavities in trabecular bone. Bone 11:241-245. 18. Parfitt AM 1983 The physiologic and clinical significance of bone histomorphometric data. In Recker RR (ed.) Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, FL, 1983, pp. 143-223. 19. Parisien M, Silverberg S, Shane E, de la Cruz L, Lindsay R, Bilezikian J , Dempster D 1990 The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous bone structure. J Clin Endocrinol Metab 70(4):930938. 20. Mosekilde L 1988 Age-related changes in vertebral trabecular bone architecture assessed by a new method. Bone 9:247250. 21. Kleerekoper M, Villanueva AR, Stanciu J , Rao DS, Parfitt AM 1985 The role of three-dimensional trabecular microstructure in the pathogenesis of vertebral compression fractures. Calcif Tissue Int 37594-597.
Address reprint requests to: Dr. M . C . de Vernejoul INSERM Unit& 18 Centre Viggo Petersen Hdpital Lariboisier 6 rue Guy Patin 75010 Paris, France Received for publication June 25, 1990; in revised form March 25, 1991; accepted March 28, 1991.
Relationship Between the Number of Resorbing Cells and the Amount Resorbed in Metabolic Bone Disorders MARTINE COHEN-SOLAL, CAROLINE MORIEUX, and MARIE-CHRISTINE DE VERNEJOUL
ABSTRACT The relationship between bone-resorbing cells, assessed by the presence of tartrate-resistant acid phosphatases (TRAP) and morphologic indices of bone resorption, was determined in 29 osteoporotic patients (14 postmenopausal females and 15 males) and 15 dialyzed patients. The number of TRAP-positive cells per unit of cancellous bone area (N.Oc/B.Ar) was higher in dialyzed patients than in those with osteoporosis (16.8 k 15.3 versus 4.95 + 2.86, p < 0.05). The amount of bone resorbed at the basic multicellular unit level was estimated by calculating eroded area containing TRAP cells per bone area (E.Ar+/BA). This novel parameter was similar in dialyzed and in osteoporotic patients (41,700 k 28,400 versus 32,300 k 24,600). In contrast, trabecular spacing (Tb.Sp) was identical in both metabolic bone diseases. Trabecular width (169 + 38 versus 127 k 32 pm, p < 0.05) and bone area were higher in dialyzed than in osteoporotic patients. N.Oc/B.Ar was significantly related to E.Ar+/BA in dialyzed ( r = 0.76, p < 0.05) but not in osteoporotic patients. Tb.Sp was significantly correlated to N.Oc/B.Ar and to the number of TRAP-positive cell nuclei per B.Ar ( r = 0.44, p < 0.05) in osteoporotic but not in dialyzed patients. This last result shows that in overt osteoporosis with thin trabeculae, trabecular spacing is related to the number of resorbing cells. In contrast, the spacing of thick trabeculae in dialysis osteodystrophy is not dependent on the number of osteoclasts.
INTRODUCTION
B
is the end result of the balance between osteoblast and osteoclast activity at the tissue level. The relationship between bone cell number and other morphologic indices is therefore an important point in understanding the mechanisms of bone loss. During aging and in osteoporosis, cancellous indices related to bone formation are diminished: trabecular and wall thickness decrease with age and are further decreased in osteoporotic patients The imcompared to age-matched control paired bone formation in osteoporosis is associated with changes in cell number: we observed a decreased osteoblast that was associated with a decreased in vitro proliferating capacity of osteoblasts isolated from bone biopsies. 1 5 ) The increase in trabecular spacing, a structural change occurring during aging and osteoporosis, is believed to be a major determinant of bone and may decrease O N E MASS
bone strength. It has been related to osteoclastic trabecular perforation during the years following the menopause. 1 2 ) To test the hypothesis that the osteoclast activity plays a role in our population with overt osteoporosis, we examined the relationship between the resorbing cells identified by the presence of cytoplasmic tartrate-resistant acid phosphatase (TRAP) and morphologic indices of cancellous bone. We studied three different metabolic bone states: ( 1 ) renal osteodystrophy, a condition not associated with bone loss"); (2) osteoporosis; and (3) normal subjects. Our data show that, in the osteoporotic patients only, trabecular spacing is related to the number of resorbing cells and resorbing cell nuclei.
PATIENTS AND METHODS
Patients and control subjects We studied three groups of patients. The first group consisted of 29 osteoporotic patients who had a bone bi-
INSERM Unite 18, Centre Viggo Petersen, Hapita1 Lariboisiere, Paris, France. 915
COHEN-SOLAL ET AL.
916
opsy for evaluation of bone status. All presented one or more vertebral compression fractures on spinal x-ray. They were divided into two groups: 14 women (mean age 68.7 k 8 years) who had undergone menopause 10 years earlier and 15 men (mean age 56 f 12) with idiopathic osteoporosis but without endocrine disease or corticosteroid therapy. However, 6 of them gave a history of chronic alcoholism ( > 80 g/day) or tobacco ( > 20 packs a year) intoxication. The second group consisted of 15 dialyzed patients (mean age 60 k 8 years, mean duration of dialysis 114 f 77 months) for whom the bone biopsy was performed to assess a possible secondary hyperparathyroidism. A total of 21 healthy young men (mean age 32.8 + 4.7), used as controls (third group), were the subjects previously reported in the study of Palle et a1.I') The stained sections were kindly provided by Dr. Alexandre (Saint-Etienne, France).
Methods The transiliac bone biopsies were embedded in methyl methacrylate at low temperature with slight modifications of a method previously d e ~ c r i b e d . ' ~Sections ' were cut with a heavy microtome (type Jung K). The sections were stained using naphthol ASTR phosphate as a substrate for tartrate-resistant acid phosphatases1Io1and counterstained with toluidine blue, pH 2.4. All the histomorphometric measurements were performed on two or three distant sections using an objective eyepiece Zeiss Intergratte plate I1 and a semiautomatic image analyzer (ASM, Leitz, Germany). The measurements were performed on total cancellous bone, and results are expressed in the second dimension according to ASBMR standardized nomenclature. ( I L 1 The bone microstructural indices were calculated from the perimeter and area measurements according to Parfitt et al."': Bone area B.Ar/T.Ar To
=
Trabecular width Tb.Wi, Fm B.Ar/B.Pm
B.Ar/T.Ar x 100 =
2000/1.199
Trabecular number Tb.Nb/mm' B. Pm/T. Ar Trabecular spacing Tb.Sp, pm (T.Ar - B.Ar)/B.Pm
=
=
X
1.199/2 x
rotic and dialyzed patients; the count was not possible in the normal subjects because of a slight difference in the technical procedure. Oc.Pm/B.Pm: osteoclastic perimeter expressed as a percentage of total cancellous perimeter. E.Pm/B.Pm: eroded perimeter expressed as a percentage of total cancellous perimeter.
To assess the amount of bone resorbed, we performed several measurements on every eroded cavity (E) containing TRAP cells (+) or without TRAP cells (-). The cavities containing osteoid were excluded. For each cavity were measured The length of the previous quiescent surface by eye reconstruction following the shape of the trabeculae on either side (E.length- or E.length+). The mean depth (E.De- or E.De+) by measuring the orthogonal distances between the reconstructed length and the base of the cavity at equidistant points indicated by the line of the eyepiece graticule (Fig. 1). The eroded area (E.Ar- or E.Ar+) was calculated indirectly by multiplying the length of the reconstructed line and the mean depth: E.Ar-
=
E.length- x E.De-
E.Ar+
=
E.length+ x E.De'
The eroded area was also expressed as an index using the bone area as referent: E.Ar-/B.Ar
=
E.Ar-/(B.Ar/T.Ar)
E.Ar'/B.Ar
=
E.Ar+/(B.Ar/T.Ar)
Reproducibility and statistical analysis The intraobserver reproducibility of our method was assessed by remeasuring six bone biopsies at an interval of 1 month by the same observer. The same biopsies were measured by another observer trained by the first for interobserver reproducibility. The coefficients of variation were calculated according to Compston et aL1''' The results are expressed as means and standard deviations. The unpaired Student's f-test was used to test the significance of differences between groups. Correlations were assessed using linear regression analysis.
2000/1.199 x
The following measurements were performed at a x 160 magnification:
N.oc/T.Ar and N.oc/B.Ar: number O f osteoclasts per m m 2 tissue area and per m m 2 bone calculated by counting only TRAP-positive cells in cancellous area and using the tissue and bone area as referents. N.Oc.Nc/T.Ar and N.Oc.Nc/B.Ar: number of osteoclast nuclei per mm2 tissue area and per mm' bone area, calculated by counting the number of osteoclast nuclei in cancellous area and using the tissue and bone area as referents. Those variables were counted only for the osteopo-
RESULTS The results of the intraobserver reproducibility show a coefficient of variation of 9.68% for E.length-, 10.9% for E.length+, 12% for E.De+, and 13% for E.De-. For the interobserver reproducibility, the coefficient of variation was 14% for E.length-, 14% for E.length+, 15% for E.De+, and 10% for E.De. The microstructural indices (Table 1) showed that there were no differences in any variables between osteoporotic males and females; the data were therefore pooled. As expected, all the microstructural indices were lower in the
ASSESSMENT OF BONE RESORPTION
917
osteoporotic group than in the controls except trabecular spacing, which was increased. Compared to controls, patients with renal osteodystrophy had decreased trabecular number and increased trabecular spacing. Bone area, trabecular width, and trabecular number were higher in dialyzed patients than in osteoporotics, whereas trabecular spacing was not different. The resorption indices are reported in Table 2. The number of osteoclasts and osteoclast nuclei number were significantly higher in dialyzed compared to osteoporotic patients, whether expressed per tissue or bone area. Reflecting the heterogeneity in the renal osteodystrophy group, the range of osteoclast number values is wide in dialyzed patients: some patients had values as low as those in osteoporotics, whereas others had fivefold higher values. In the osteoporotic group, the slightly higher osteoclast number value in females compared to males was not significantly different. When osteoclast and osteoclast nuclei counts were expressed using bone area as referent, the women had higher osteoclast indices than the men. In both diseases, the Pumber of osteoclasts was increased compared to controls. E.De- compared to E.De' or E.Ar- compared to E.Ar+ was never significantly different in any of the three groups.
In the dialyzed patients, the erosion depth with osteoclasts was higher so that the absolute value of E.Ar+ is increased compared to that in osteoporotics and controls. When expressed with B.Ar as referent, however, there is no difference because of a lower bone area in osteoporotics. The eroded area with osteoclasts (E.Ar+/B.Ar) was significantly increased in osteoporotic patients compared to controls, also due to a smaller bone area, whereas the eroded and osteoclast perimeters of osteoporotics and controls were similar. To correlate cell or nuclei number and the other variables, only osteoclast number using bone area as referent was considered. In patients with renal osteodystrophy, the eroded area with osteoclasts was correlated with the osteoclast nuclei number ( r = 0.69, p < 0.01) and osteoclast number ( r = 0.76, p < 0.01; Fig. 2). However, the E.Ar-/ B.Ar and osteoclast indices were not correlated with trabecular width, number, or spacing. In contrast, in the control and osteoporotic groups, we could not find a relationship between eroded area with osteoclasts and the osteoclast indices. However, in the osteoporotics only, there was a positive correlation between Oc.Nb/B.Ar and Oc.Nc.Nb/B.Ar with the trabecular spacing ( r = 0.37, p < 0.05; r = 0.44, p = 0.01; Fig. 3).
DISCUSSION
FIG. 1. M~~~~~~~~~~ of eroded area. ~h~ previous quiescent surface was reconstructed following the curvature of the trabeculae. The depth was measured as the orthogonal distance between this line and the base of the cavity.
Renal osteodystrophy and osteoporosis are two markedly different disorders. In dialyzed patients parathyroid hormone is the main factor regulating bone resorption.' 1 3 ) In contrast, this calcitropic hormone is not the main factor regulating osteoclast number or activity in osteoporosis. The relationship between resorbing cells and the amount of bone resorbed in both circumstances could therefore lead to different data. Osteoclasts can better be determined by histochemical identification of TRAP-positive cells. Indeed, multinuclearity is an uncertain criterion, since it has been shown that most osteoclasts have no nuclei visible on the section or contain only one nuclei.f14)In this study, the
INDICES TABLE1. STRUCTURAL Osteoporotic patients Index B.Ar/T.Ar, % T b Width, pm Tb.Nb, mm' Tb.Sp, pm
Controls (n = 21) 28.6 159 1.66 449
f f f f
6 36.9 0.22 83
Females (n = 14)
Males (n = 15)
Combined (n = 29)
Dialyzed patienls (n = 15)
13.5 + 4.6 122 5 36 1.13 + 0.3 814 + 210
17.2 f 5.3 131 + 27 1.31 k 0.3 697 f 243
15.3 k 5.2a 127 k 32a 1.22 + 0.35a 757 f 230a
22.7 k 12b 169 f 38b.C 1.35 0.56b 660 k 250d ~~
a C ~ n t rversus ~ l ~ combined osteoporotic patients, p < 0.01. Wombined osteoporotic versus dialyzed patients, p < 0.05. 'Combined osteoporotic versus dialyzed patients, p < 0.01. dControls versus dialyzed patients, p < 0.01.
COHEN-SOLAL ET AL.
918 TABLE2. RESULTSOF
THE
RESORPTION INDICES
Osteoporot ic patients Controls (n = 21) N.Oc/T.Ar, mm-’ N. NcOc/T.Ar, mm-’ N.Oc/B.Ar, mm-* N.OcNc/B.Ar, mm-’ Oc.B.Pm/B.Pm, Vo E.B.Pm/B.Pm, Vo
0.79 f 0.37
E.De-, pm E.De+, pm E.length-, pm E.length+, pm E.Ar-, pm’ E.Ar+, pm’ E.Ar-/B.Ar, per lo00 E.Ar’/B.Ar, per lo00
23.6 f 22.5 f 223 f 247 f 5300 f 5600 f 1.93 f 2.01 f
-
2.91 f 1.56 -
1.69 f 0.73 3.77 f 1.46 6.9 4.1 55 44 2550 1670 1.14 6.54
Females (n = 14)
0.77 0.95 6.08 7.81 1.5 4.42
Males (n = IS)
Combined (n = 29)
f f f f f f
0.36 0.45 3.2 3.78 0.9 1.15
0.58 0.67 3.89 4.46 1.1 3.22
f f f f f f
0.27 0.38 2.1~ 3.le 0.5~ 0.93e
0.67 0.81 4.95 6.08 1.29 3.87
20.7 f 19.6 f 177 f 208 f 3740 f 4490 f 2.83 f 3.59 k
3.78 5.9 62 95 1620 3240
20.6 20.7 189 217 3910 4570 2.55 2.85
f f f f f f f
2.85 3.5 78 89 1730 2120 1.5 1.43f
20.7 f 3.31f 20.1 f 4.87 183 f 69f 212 f 91 3820 f 1650 4530 f 2710 2.69 f 1.27f 3.23 k 2.46f
104
3.15
k
f f f f f f
0.33 0.43 2.86d 3.79 0.79 1.52
Dialyzed patients (n = 15)
3.58 f 5.85 f 16.5 f 25.3 f 3 f 6.74 f
3.53a 6.74b 14.9a 24.6b 2.08b 2.3P
30 f 16.8~ 46.1 f 22.7a 157 f 7 9 182 57h 5110 f 2910 8430 f 5240i 2.94 f 1.09j 4.17 f 2.831
*
dDialyzed patients versus controls and combined osteoporotic versus dialyzed patients, p < 0.01. Wombined osteoporotic versus dialyzed patients, p < 0.01. LFemale versus male osteoporotic patients, p < 0.05. C o m b i n e d osteoporotic patients versus controls, p < 0.01. ‘Female versus male osteoporotic patients, p < 0.01. ‘Combined osteoporotic patients versus controls, p < 0.05. wDialyzed patients versus controls, p < 0.05, and combined osteoporotic versus dialyzed patients, p < 0.01 hDialyzed patients versus controls, p < 0.01. ]Dialyzed patients versus controls and combined osteoporotic versus dialyzed patients, p < 0.05. ]Dialyzed patients versus controls, p < 0.05.
amount of bone resorbed at the basic multicellular unit (BMU) level as evaluated by an original method taking into account not only the depth of the resorption cavity(8.1S) but also its length. Because the osteoclast is a highly motile the length of the lacunae also reflects the activity of the cell. Moreover, the eroded and osteoclast surfaces seem to be insufficient variables to assess the resorption: in the present study, for example, the E.Ar+ or E.Ar‘/B.Ar was higher in osteoporotics, despite the absence of difference in osteoclast or eroded perimeters. Such an attempt to measure resorption cavity has recently also been made by Garrahan et al.[17’The method we describe is very similar and indeed values observed concerning mean depth, length, and absolute area of the resorption cavities in normal subjects are very close in both studies. The slight difference may be due to automatic direct measurement[”) compared to calculated index in our study. However, reproducibility was similar in both methods. We separated values obtained in cavities either with or without TRAP-positive cells. We expected values of the mean depth and area of cavities devoid of TRAP cells to be higher because the resorption process is supposed to be achieved in these “reversal” This was not the case, neither in controls nor in any metabolic bone diseases. This might be because lacunae without TRAP cells may contain TRAP cells in another section plane. Another
possible explanation for this observation is that osteoclastic resorption may not be a continuous phenomenon during the resorption phase of the cycle: there would therefore be an “on-and-off’ phenomenon, as for formation.[18’ This would indicate that all the lacunae devoid of osteoclasts are not in the reversal phase of the remodeling cycle. In patients with renal osteodystrophy, there was a clear relationship between osteoclast number and eroded area with osteoclasts. This result suggests that, at the basic multicellular unit level, osteoclast activity is dependent on osteoclast number. Eroded area without osteoclasts was not correlated to osteoclast number. This significant relationship between osteoclast number and eroded area was not observed in osteoporosis. Taking into account osteoclast nuclei instead of osteoclast number did not give a significant relationship, either. This negative result may be due to variable osteoclastic activity among patients or to regulation by two different factors of osteoclast number and activity in osteoporosis. Structural indices of bone architecture reflect bone cell activity at the tissue level. They are useful indices of threedimensional microstructures and have shed new light on the mechanism of bone loss. In our study, bone area and trabecular width were higher in dialyzed patients whereas trabecular spacing was identical in both groups. Trabecular thickness has been shown to reflect bone formation and
919
ASSESSMENT OF BONE RESORPTION
E.Ar +I B.Ar
10
20
30
40
50
60
70
N.Od B.Ar (/ mm2)
FIG. 2. Relationship between osteoclast numberlbone area (N.Oc/B.Ar) and eroded area with osteoclast/bone area (E.Ar'/B.Ar) in 15 dialyzed patients: r = 0.76, p < 0.01).
4ooo1
':
**lo
,
2000
0
10
,
higher in renal osteodystrophy than in osteoporosis, but formation is also higher. This is due to the dual effect of parathyroid hormone in both formation and resorption. This suggests that the balance between resorption and formation is preserved in secondary hyperparathyroidism, as well as in primary hyperparathyroidism.l") Thick trabeculae cannot be perforated even in the presence of a large number of osteoclasts; indeed, in such circumstances trabecular spacing does not reflect the number of resorptive cell at the tissue level. During aging and in osteoporosis, Parfitt et al.'') have shown that bone loss is primarily caused by the complete loss of trabecular plates, which increase separation between remainders. In osteoporosis, mainly in female patients, trabecular spacing was further increased compared to that in age-matched controls.I2) I t has been proposed that trabecular separation could be explained by intense resorptive activity at the time of menopause." ' ) Because of the lack of an age- and sex-matched control group for the osteoporotic patients in this study, we cannot conclude whether it is related to increased erosion depth or increased resorption sites. Our data are in agreement with those of other studies showing thin trabeculae in osteoporosis'' '01 related to decreased bone formation. The close relationship observed between osteoclast number and trabecular separation suggests that trabecular separation by increased osteoclast number is not a phenomenon limited to the years following the menopause but a process still occurring in older female and male osteoporotic patients. The implication for osteoporosis treatment of such a structural consequence of increased osteoclast number might improve therapeutic efficacy in osteoporosis as increased trabecular spacing has been shown to be an important risk factor for fractures."" ' I )
ACKNOWLEDGMENTS
20
N.Oc.Nc/ B.Ar (/ mm2)
FIG. 3. Relationship between osteoclast nuclei number/ bone area (N.Oc.Nb/B.Ar) and trabecular spacing in 29 osteooorotic oatients: r = 0.44. D < 0.05).
The authors are fully grateful to Dr. Parfitt for reviewing the manuscript and to Drs. Alexandre and Chappard for providing the slides.
REFERENCES is related in normal subjects to wall thickness.IJ) Trabecular spacing was comparable in dialyzed and osteoporotic patients; however, osteoclast number was higher in dialyzed patients. It is not likely that this result is due to lower osteoclast resorption activity in dialyzed rather than in osteoporotic patients since the absolute value of eroded area with osteoclasts was higher in dialyzed patients than in osteoporosis. However, because the N .Oc/B.Ar is fivefold higher, we also expected to find E.Ar'/BA fivefold higher; it cannot be excluded that the osteoclast activity is slightly lower in renal osteodystrophy. An identical value of trabecular spacing in the face of a higher osteoclast number can be explained by a greater trabecular width in renal osteodystrophy. Moreover, osteoclastic resorption is
I . Lips P , Courpron P , Meunier P J 1978 Mean wall thickness of trabecular bone packets in the human iliac crest: Changes with age. Calcif Tissue Res 26:13-17. 2. Parfitt C , Mathews C H E , Villanueva AR, Kleerekoper M , Frame B, Rao DS 1983 Relationships between surface, volume, and thickness of iliac trabecular bone i n aging and in osteoporosis. J Clin Invest 73:1396- 1409. 3. Aaron J E , Makins NB. Sagreiya K 1987 The microanatomy of trabecular bone loss in normal aging men and women. Clin Orthop 215:260-271. 4. Garcia-Carrasco M, dc Vernejoul MC, Sterkers Y , Morieux C , Kuntz D, Miravet L 1989 Decreased bone formation in osteoporotic patients compared to age matched control. Calcif Tissue Int 44:173-175. 5. Marie P J , Sabbagh A, de Vernejoul MC, Lomri A 1989 Osteoblastic cells isolated from posf-menopausal osteopo-
COHEN-SOLAL ET AL.
920
6.
7.
8.
9.
10.
11.
12.
13.
14.
rotic women: Relationships between osteocalcin and DNA synthesis in vitro and histomorphometric indices of bone formation. J Clin Endocrinol Metab 69:272-280. Weinstein RS, Huton MS 1987 Decreased trabecular width and increased trabecular spacing contribute to bone loss with aging. Bone 8:137-142. de Vernejoul MC, Marchais S, London G, Bielakoff J , Morieux C , Miravet L 1985 Increased in stainable bone aluminum after subtotal parathyroidectomy in dialysed patients. Kidney Int 27:785-791. Palle S, Chappard D, Vico L, Riffat G, Alexandre C 1989 Evaluation of the osteoclastic population in iliac crest biopsies from 36 normal subjects: A histoenzymologic and histomorphometric study. J Bone Miner Res 4:501-505. Chappard D, Palle S, Alexandre C , Vico L, Riffat G 1987 Bone embedding in pure methyl methacrylate at low temperature preserves enzyme activities. Acta Histochem (Jena) 81: 183- 190. Baron R, Vignery A, Neff L, Silverglate A, Santa Maria A 1983 Processing of undecalcified bone specimens for bone histomorphometry. In Recker RR (ed.) Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, FL, pp. 13-35. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H. Meunier P , Ott SM, Recker RR 1987 Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 2(6):595-610. Compston JE, Vedi S, Stellon AJ 1986 Inter-observer and extraobserver variation in bone histomorphometry. Calcif Tissue Int 38:67-70. de Vernejoul MC, Kuntz D, Miravet L, Gueris J , Ryckewaert A 1981 Bone histomorphometry in hemodialysed patients. Metab Bone Dis 3:175-169. Baron R, Neff L, Tran Van P , Nefussi JR, Vignery A 1986 Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am J Pathol 122:363-378.
15. Eriksen EF, Mosekilde L, Melsen F 1985 Trabecular bone resorption depth decreases with age: Differences between normal males and females. Bone 6:141-146. 16. Kanehisa J , Heersche JNM 1988 Osteoclastic bone resorption: In vitro analysis of the rate of resorption and migration of individual osteoclasts. Bone 9:73-79. 17. Garrahan NJ, Croucher PI, Compston J E 1990 A computerised technique for the quantitative assessment of resorption cavities in trabecular bone. Bone 11:241-245. 18. Parfitt AM 1983 The physiologic and clinical significance of bone histomorphometric data. In Recker RR (ed.) Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, FL, 1983, pp. 143-223. 19. Parisien M, Silverberg S, Shane E, de la Cruz L, Lindsay R, Bilezikian J , Dempster D 1990 The histomorphometry of bone in primary hyperparathyroidism: Preservation of cancellous bone structure. J Clin Endocrinol Metab 70(4):930938. 20. Mosekilde L 1988 Age-related changes in vertebral trabecular bone architecture assessed by a new method. Bone 9:247250. 21. Kleerekoper M, Villanueva AR, Stanciu J , Rao DS, Parfitt AM 1985 The role of three-dimensional trabecular microstructure in the pathogenesis of vertebral compression fractures. Calcif Tissue Int 37594-597.
Address reprint requests to: Dr. M . C . de Vernejoul INSERM Unit& 18 Centre Viggo Petersen Hdpital Lariboisier 6 rue Guy Patin 75010 Paris, France Received for publication June 25, 1990; in revised form March 25, 1991; accepted March 28, 1991.
