Bone andMineral,
ll( 1990)247-259
Elsevier
247
BAM 00320
L.
. Mordon’ and
‘Department of Rheumatology, The General Infirmary, Leeds LSI 3EX, England and ‘General Clinical Research Center, Indiana UniversityHospital, E/534, 926, W. Michigan Street, Indianapolis, IN 46202-5250, USA
(Received 20 December 1989) (Accepted 18 July 1990)
Iliac crest bone histomorphometry, plasma and urine biochemistry and clinical history were examined in 78 unselected patients (68 women, i8 men) at the time of femoral fracture. Histological abnormalities occurred in 56 of the 78 biopsies. The commonest of these was a low bone volume of less than 15% which, irrespective of other abnormal histological features, was present in 37 of the biopsies. On the basis of the histomorphometry, patients could be classified into four main groups. Normal histomorphometry (bone volume >15%, osteoid surfaces 60%) was present in 22 patients, 23 had osteoporosis as the only abnormality (bone volume ~15%. osteoid surface 60%), nine had osteomaiacia (osteoid surfaces >24%, mineralising surface 13 pm) and 13 had decreased mineralising surfaces. Of the remainder, five had increased osteoid surface and six had insufficient osteoid to assess mineralising surface. Plasma and urine biochemistry in the four groups showed that, compared to age-matched controls,, all groups had reduced plasma albumin. In comparison to the group with normal histomorphometry, patients with osteoporosis had a higher plasma calcium (P < O.OI), tubular reabsorption of calcium (P < 0.05) and plasma vitamin D binding protein (P < 0.01); patients with ostewmalacia had a higher plasma creatinine (P < 0.02) and parathyroid hormone (P < 0.02) and lower plasma 24.25dihydroxyvitamin D (P < 0.02), urinary calcium/ creatinine ratio (P < 0.02) and tubular reabsorption of phosphate (P < 0.02). The biochemistry in patients with decreased mineralising surface was no different from patients with a normal biopsy. The prevalence of both osteoporosis and osteomalacia increased with age and, in subjects over the age of 90, osteoporosis occurred in 71% of patients and osteomalacia occurred in 29% of patients. The osteomalacic group were significantly older than the other three groups (P C 0.05). The histomorphometry did not relate to the site of fracture (subcapital or intertrochanteric). A history of stroke, gastrectomy, rheumatoid arthritis, steroid treatment, thyroid disease, alcohol abuse and anticonvulsant therapy was present in patients with femoral fracture but did not relate to any particular histomorphometric classification.
Key words: Osteomalacia; Osteoporosis; Bone histomorphometry; Femoral neck fracture; 1,25(OH),D
Correspondence to: Munro Peacock, M.D. General Clinical Research Center, Indiana University Hospital, N-534,926 W. Michigan Street, Indianapolis, IN 46202-5250,USA. 0169~600!?/90/$03.50@1990Elsevier Science Publishers B.V. (Biomedical Division)
248 Introduction
Femoral neck fracture is the commonest fracture in people over the age of 65 [l] causing considerable mortality and morbidity [2]. In populations of Northern European descent, femoral neck fracture is mainly a disease of elderly women [3-61. Moreover, the incidence of this fracture is increasing over and above the increase in the elderly population, especially in women over the age of 75, the reason for which is not clear. Although many studies have looked at the roles played by osteomalacia and osteoporosis in the pathogenesis of this fracture, their prevalence and relative contributions to the fracture are still disputed. Much of the controversy arises from differences in populations studied, and, particularly in the case of osteomalacia, from different biochemical, radiological and histomorphometric criteria used for diagnosis. The prevalence of osteomalacia in the femoral neck fracture population is of more than academic interest. Whilst advances are being made in the prevention of osteoporosis [7], treatment of established osteoporosis remains difficult [8]. On the other hand, osteomalacia, if detected, can usually be prevented and treated. The aims of our study were to examine bone histomorphometry and its relation to plasma biochemistry and vitamin D metabolites in the femoral neck fracture population to determine whether any clinical or biochemical criteria could. at the time of fracture, identify patients with histologica abnormalities, particularly those of osteomalacia.
Materials and Methods
Two hospitals in Leeds receive unselectively all patients who sustain a femoral fracture. Femoral fracture patients are admitted in rotation by four orthopedic teams and all patients who were treated surgically by one of the four teams were considered for study. Sixty-eight women, aged 52-99 (mean 78) years, and 10 men, aged 45-92 (mean 77) years, admitted to one of the hospitals with femoral neck fracture from March 1984 to February 1986, were studied. During this period, a total of 419 patients (329 women and 90 men) were admitted with femoral neck fracture and 292 of these subsequently received an operation. Our sample is, therefore, unselected and represents 19% of the fracture population admitted and 27% of those operated on for this condition. All patients were of Caucasian origin. Patients were recruited evenly over a 2-year period to exclude any seasonal variation. Patients with subtrochanteric fractures and those known to have malignant disease were excluded from the study. Fracture patients were classified radiologically into two groups, those with subcapital and transcervical fractures, and those with basal cervical and intertrochanteric fractures. The biopsy was performed at the time of surgery which was performed a median of 1 day after fracture (range O-12 days). Informed consent was obtained from all subjects.
249 Methods
Transilial bone biopsies were obtained 1 inch below and behind the anterior superior iliac spine using an 8 mm Bordier trephine. The bone biopsies were fixed in 10% phosphate-buffered formalin for 48 h, dehydrated through graded alcohols, cleared in xylene, embedded in methylacrylate and left to polymerise until hard enough for sectioning. Between 30 and 48 longitudinal sections IO-pm thick were cut from each biopsy using an LKB 2258 PMV heavy duty microtome. Two-thirds of the sections were stained using Goldner to demonstrate osteoid surfaces and one-third in toluidine blue to demonstrate mineralising surfaces. The following measurements were performed on cancellous bone. Bone volume (SWTV) and osteoid volume (OVln/)
Bone volume and osteoid volume were expressed as a percentage of the total tissue volume. The amount of cancellous bone per unit area was determined on 64 fields, four fields on each of 16 sections. Each field was examined using a 25point Zeiss integrating eyepiece and a magnification of ~50 and a point on the grid coinciding with a trabecula was scored as a hit. The number of hits in the four fields of each section was taken as a measure of the percentages of bone in that section. The mean value of the 16 sections was expressed as a percentage volume of bony tissue, assuming the biopsy to be homogenous. In calculating osteoid volume, the number of hits in the four fields of each section coinciding with an ostcoid seam was taken as a measure of the percentage of osteoid in that section. The normal range of BV/TV was defined as greater than 15% [9,10] and that of OV/TV less than 1%. Osteoid surface (OSJBS) and eroded surface (ESIBS)
Osteoid and eroded surfaces were expressed as a percentage of total bone surface. To assess the surface extent of osteoid and eroded surface, a line sampling technique was used em loying the same integrating eyepiece and number of fields and sections, at a magnification of x80. All intersections between the grid lines and trabecular surfaces were counted in each field and the proportion of these intersections coinciding with osteoid or eroded surfaces counted. The results were expressed as a percentage of trabecular surface covered in osteoid or eroded and the normal range for OWBS was taken as less than 24% and for ES/BS 15
12.5 + 1.5
20.0 11.1 19.4 14.6 21.3
BVm + + f f f
0.5 0.02 0.77 0.11 0.33
0.0 cl
0.63 + 0.4
0.25 0.06 2.4 0.20 0.67
OVA’V (%)
(-) 4-13
12.0 z!L0
(-) (-) 20.9 zk 1.4 (-) 12.4 f 1.9
OW @m)
1.1 0.7 2.9 1.0 9.0
8.0 + 1.6 Cl2
f + k f f
0.4 k 0.2 ~24
9.0 8.9 11.9 8.6 23.0
7.5 + 1.5
f 1.3 f 0.7 f 9.7 + 2.7 +. 0.7
ESiBS (%)
36.5 + 6.5
7.7 5.3 53.3 8.7 25.3
OYBS (%)
Results are expressed as means k SEM, the number of men (M) in each group is indicated and variables not measured are shown as (-).
1M 4M 2M
22 23 9 13 3
Normal Osteoporosis Osteomalacia Decreased mineralising surface Increased osteoid surface Increased osteoid surface, decreased mineralising surface, osteoporosis Insufficient osteoid to assess mineralising surface Control range
Sex
N
and age in 78 patients with femoral neck fracture
Classification
Bone histomorphometry
Table 1
+ + + f +
2.4 2.1 7.7 2.6 4.2
(-) >60
35.5 f 5.5
74.9 76.5 36.0 50.6 67.7
MS/OS (%)
252
ranges given [9,10] in Table 1,22 (31%) femoral fracture patients had normal bone histdogy and 23 (32%) had as the only abnormality a bone volume less than 15% and, by definition, were osteoporotic. Osteomalacia, defined as osteoid surface greater than 24%, osteoid width greater than 13 pm and mineralising surface less than 60% [23], was present in nine patients (12%), two of whom also had a bone volume less than 15%. Thirteen patients (18%), had decreased mineralising surfaces, 7 (10%) of whom also had a bone volume less than 15%. Three patients (4%) had increased osteoid surfaces with normal mineralising surfaces, including two with increased osteoid width. Two patients (3%) had increased osteoid surface, decreased mineralising surface but with normal osteoid width and were osteoporotic. In six (8%) of the 78 biopsies there was insufficient osteqid pressnt to assess the mineralising surface. Three of these had bone volume less thzn 1.5t%,and thus of the 78 patients biopsied, 37 (47%) had a bone volume less than 15% and by definition had osteoporosis. Plasma and urine biochemistry in relation to bone histomorphometry (Table 2) For purposes of comparison four groups were defined according to histomorpho-
metric criteria and classified as; normal, osteoporosis, osteomalacia and decreased mineralising surface. The number of subjects in the groups with increased osteoid surface, decreased mineralising surface and normal osteoid width, and increased osteoid surface and normal mineralising surface were too small to analyse statistically. As compared to a group of aged-matched controls, plasma albumin was decreased in all four groups of patients with femoral fracture. Plasma creatinine was also low in all but the osteomalacic r,roup who had a significantly increased concentration (P c 0.001). The vitamin D metabolites were normal in all groups except for the group with decreased mineralisation who had a significant increase in total (P < 0.01) and free (P < 0.05) 1,25(OH),D. Plasma calcium was low in the groups with normal and with decreased mineralisation but parathyroid hormone was in the elderly control range in all groups except the osteomalacics who had a significantly increased concentration (P c 0.001). Plasma phosphate and tubular reabsorption of phosphate was increased in all but the osteomalacic group who had decreased values. As compared to the group with normal histology, patients with osteoporosis had a higher plasma calcium (P < 0.01) plasma DBP (P < O.Ol), and tubular reabsorption of calcium (fp < 0.05). Patients with osteomalacia had higher plasma creatinine (P c 0.02) and PTH (P < 0.02), and lower calciumkreatinine ratio (P < 0.02), TmP (P < 0.02) and plasma 24,25(OH),D (P < 0.02). There were no significant differences in any of the biochemical variables measured between patients with decreased mineralising surfaces alone and those with normal bone histology. Osteoporosis and osteomalacia in relation to age in femoral fracture patients (Fig. I)
The osteomalacic group was significantly older than the other three groups (P < 0.05) (Table 1). Osteomalacia was absent in the 14 patients younger than age 70 but present in 5% of the patients in the 70-79 age group, in 17% in the 80-89 age group
253
Plasma and urine biochemistry
Normal
in patients with femoral neck fracture classified
Osteoporosis
Osteomalacia
Decreased
Elderly
mineralking
controls
surface
Age (years)
79.0+2.2 (II=)‘)
69-93 (n=23)
Plasma CiIlciUm
(mmol/l) Phosphate (mmolit)
1.04+0.0# (11=X) 14.5k-l.tr
AlliitlilW
phosphatasc (LA.
’ 7’+0.02’ _.. _(fit=%)
units)
Crcatinine (qIol/l) Albumin (g/l) ‘(0H)D (nmol/l) 21.2S(OH)2D (nmolil)
1.Z(OH),D (pnK~lll) DBP (frmolil) *Free’
I .25(0H),D (pmal4) PTH
74.1-1-3.8”
97kh
01=22) 3x+ 1AI’ (11= 19) 12.9+1.3 (II- ii) 0.77z!5). I1 (If= 10) 114.3+ I-t.8 (fr= 15) fi.xto.2Y (v= 15) 1.23-tt1.17 (?I= 14) .#l7.2+49.x (If = 15)
Urine CaiCr (mol) OHPCr
9.7+o.H
(If= 19)
(mol)
39+0.x 13.5+ 1.2
93.x+5.8
1.OS~II.OX
0. 14+u.osJ
0.46~0
(tt=7)
(t1=9)
0.029+,().012 (tt
‘7)
13
0.03 1f0.004
o.u+o.
10
0.022+0.003
(a =9)
TmCa
1.Y6+0.Oh
1.83+o.M
1.92+0,004
TmP
(rF=7) 0.74+0. lo”~C
(a=9) 1. itp-I_0.05’
0.93+0.03
(n=7)
(n=9)
Results are expressed as means f. SEM. Significance between rhe group with normal hislolopy and other groups are shown as: “r” < 0.115. “p c 0.02. “P)< 0.01 and between groups and tlderly controls (371 as: dP < 0.05. ‘P 4 0.~11.‘P c 0.001.
254
Fig. 1. Bone volume in relation
to age in patients with femoral neck fracture. Patients with (0) ad ) osteomalaciaare shown.
and in 29% in the 90-99 age group. Osteoporosis was present in 43% of the patients younger than age 70, in 52% in the 70-79 age group, in 53% in the 80-89 age group and in 71% in patients over the age of 90. Ten patients were male and two of these (20%) had osteomalacia. Mean bone volume in the 61 women without osteomalacia was 15.8 + 0.8% (SE) and in the eight men without osteomalacia was 15.6 f 2.1% (SE). Bone histomorphometry in relation to type of femoral neck fracture
Of 78 femoral neck fracture patients studied, 44 (56%) had subcapital fractures and 34 (44%) had intertrochanteric fracture. There was no relationship between bone histomorphometry and the type of fracture. Previous fractures in femoral fracture patients in relation to bone histomorphometry
None of the 10 male patients had previously suffered a femoral neck or wrist fracture. Of the 68 female patients, 15 (22%) had had a previous femoral neck fracture and 13 (19%) had previously sustained a unilateral or bilateral wrist fracture. Four of the seven (57%) female patients with osteomalacia had previously fractured a femoral neck and three (43%) had previously had !i wrist fracture. Although female osteomalacic patients appeared more likely to have had a previous femoral fracture than patients with other histological diagnosis, this trend was not significant (2 with Yates correction = 3.54). Clinical history in femoral neck fracture
A history of stroke (four patients), gastrectomy (five patients), rheumatoid arthritis (six patients), steroid treatment (six patients), thyroid disease (six patients), alcohol abuse (six patients) and anticonvulsant therapy (five patients), was obtained but did not relate to the histological classification.
255
Our study shows that patients biopsied at the time of femoral fracture, thus reducing immobilisation effects, frequently have histomorphometric abnormaiities in cancellous bone. In only 31% of the patients was the biopsy normal. The most frequent abnormality was a bone volume less than 15%. In 32% of patients this occurred in the absence of any other abnormality but in the group as a whole, irrespective of other abnormalities, it occurred in 47% of patients. Osteomalacia, a failure of osteoid to calcify as it is laid down, requires double tetracycline labelling for diagnosis which is difficult to do in patients biopsied at the time of femoral fracture. However, using the coexistence of increased osteoid surface, increased osteoid width and decreased mineralising surfaces, generally accepted criteria indicating the presence of osteomalacia, we found that osteomalacia has an overall prevalence of 12%. Thus it remains a significant abnormality in patients with femoral neck fracture in Britain, and the same is probably true of Europe [24] and Elorth America [25,26]. The occu~ence of both osteomalacia and osteoporosis in our study are age-related, with osteomalacia being particularly high in those over the age of 90 years. The prevalence of osteomalacia in our study was lower than the 20% reported in a previous study from the same center [9]. In that study, however, the criteria for osteomalacia did not inciude osteoid width. Using the same criteria as in that study, the prevalence of osteomalacia in our series would increase to 15%. On the ot igher than that of 2% in a recent study from a similar geographical location to our own [27]. In the later study, osteomalacia was defined as the presence of an osteoid area greater than 5%) making comparison difficult with our study. Five patients (6%) had increased osteoid surfaces, but did not fulfill all the criteria for osteomalacia. The reason for this osteoid increase is not clear. There was no biochemical evidence of primary hyperparathyroidism or clinical history of thyroid disease. Early vitamin D depletion has been shown to cause accelerated bone remodelling and increase osteoid surfaces [28] and this is a possible explanation, particularly in the two very elderly patients who also had low mineralising surfaces and in the one patient on anticonvulsant therapy. The mean BWTV of 15.8 + 5.8 (SD) in female femoral neck fracture patients without osteomalacia was lower than the mean BWTV of 19.6 f 5.6 (SD) reported for healthy American women aged 65-74 [29], and that of 19.8 + 3.9 (SD) described for healthy British women aged 61-80 [30], but there was considerable overlap as has been found in other histomorphometric [9,31] and radiographic studies [32,33]. There were no differences in bone histomorphometry between subcapital and intertrochanteric fracttircr : as found in a previous study [9]. This finding contrasts with others which found that intertrochanteric fractures were present more often in severely osteoporotic women [X,34], and subcapital fractures predominated in osteomaiacic patients [27]. As a group the most striking biochemical abnormality in patients with femoral fracture was 5 subnormal plasma albumin. Low plasma albumin has been noted in
256 other studies [35-373 and is generally assumed to be due to poor nutrition and intercurrent illness. We think it is also related to the trauma and the surgery since 6 months after treatment the low plasma albumin reverts into the normal range [37]. As compared to young normals, increased levels of plasma PTH are a frequent finding in the elderly with [37-391 and without femoral fracture [40]. Multiple factors are probably responsible including renal failure, vitamin D deficiency and insufficiency, calcium malabsorption and poor calcium intake. In our fracture patients the plasma PTH was increased above the young normal range of 125-375 pg/ml but only in the osteomalacic group was it significantly increased from the elderly controls and it was not associated with an increase in erosive surfaces. After fracture, plasma biochemistry changes with an immediate fall in plasma calcium and albumin, and rise in plasma phosphate [41,42], and a late rise in alkaline phosphatase in the second week [43]. Plasma 1,25(OH),D metabolism is also affected by both fracture and hip surgery [37] with a failure of plasma levels to change in response to changes in plasma 25(OH)D. These changes confound the biochemical diagnosis of metabolic bone disease particularly that of osteomalacia. Although the osteomalacic patients in our series had the lowest plasma 25(OH)D levels, they were not significantly lower than those with normal bone histomorphometry. However, 24,25(OH),D was significantly reduced suggesting that the osteomalacic group had the lowest vitamin D status. It has been suggested that 24,25(OH),D plays a specific role in the mineralisation of bone and may be important in femoral fracture [44]. Others, however, have shown that 24,25(OH),D administration has no unique effect on calcium metabolism in osteomalacia [45] and that complete healing of osteomalacia occurs with 1,25(OH),D, administration in the absence of measurable levels of 24,25(OH),D and 25(OH)D [12]. The osteomalacic patients had significantly higher plasma PTH levels and alkaline phosphatase activity, lower TmP, and urine calcium indicating that in this group the overall vitamin D status had progressed from a state of insufficiency to one of true deficiency [46]. It is likely, therefore, that the low 24,25(OH),D simply represents a depression of the 24-hydroxylase by the raised PTH levels. Overali, the biochemistry is helpful in separating the osteomalacics as a group, but less so in the individual. Patients with decreased mineralising surface alone, either with or without osteoporosis, showed no difference in plasma or urine biochemistry from the patients with normal histomorphometry. The mineralising defect may represent an early stage of osteomalacia [47] as this is the first abnormality to occur in the absence of dynamic measurements using tetracycline labelling. However, this appe;irs unlikely to US since the biochemistry of this group is not different from the normal group. The mineralising surface taking up toluidine blue has been shown by some authors to correspond closely to the fraction of osteoid showing tetracycline labelling 148,491although this is disputed [50]. A decrease in the fraction of osteoid showing tetracycline labelling has been reported in patients with osteoporosis [51,52] and it is perhaps significant that over half of our patients who showed decreased mineralising surface but no change in osteoid also had osteoporosis. The decrease in mineralising surface, therefore, may be more a feature of the osteoporotic than the os-
257 teomalacia process. The main clinical condition contributing to osteoporosis and, particularly, to OSteomalacia in femoral neck fracture patients was age. In addition, over half of WOmen with osteomalacia had a previous femoral fracture, compared with a fifth of women with other histology. This may reflect the poor muscle strength and decreased cortical bone [26] of osteomalacia, together with the tendency of Looser’s zones to occur bilaterally. On the other hand, femoral fracture impairs mobility which may precipitate osteomalacia by reducing sunlight exposure. It appears equally likely, therefore, that femoral fracture itself is a risk factor for osteomalacia. It is well known that renal function declines with age [53] and previous work has shown that elderly women with a plasma creatinine over 100,~molll have a GFR of ~50 mllmin, reduced calcium absorption and decreased plasma 1,25(0 ]39]* It is not surprising, tfierefore, that the group with osteomalacia showed the greatest impairment of renal function. surprising is that despite t ey were still able to maintain plasma 1,25( levels in the normal range, albeit with evidence of secondary hyperparathyroidism. The results of this study in femoral neck fracture patients emphasise the high prevalence of abnormalities in bone histomorphometry, the commonest of which is osteoporosis, Osteomalacia is also a significant problem and in the individual patient difficult to diagnose biochemically at the time of fracture. The prevalence of botil osteoporosis and articularly osteomalacia increase with age suggesting that patients with femoral neck fracture as a group have vitamin D insufficiency and calcium malabsorption, which in older patients readily progresses into one of true deficiency resulting in a failure of the osleoid to calcify [54].
We wish to gratefully acknowledge the assistance of the orthopedic surgeons, M.J. Abberton, Mr E.B. Longton, Mr M.A. Nelson and Mr F.F. Silk and their staff, particularly Mr Austen Wynne under whose care the patients were admitted and their bone biopsied. We are also grateful to Mr G.A. Taylor, Dr J. Storer and Dr R.W. Marshall for the biochemical measurements, Dr David Marshall for advice on the statistics Dr J. Aaron and Mr J. Thompson for advice with the histological techniques, and Mrs M. Cromer and Mrs D. Holdsworth for typing the manuscript.
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259 30 Vedi S, Compston JE, Webb A, Tighe JR. Histomorphometric analysis of bone biopsies from tne iliac crest of normal British subjects. Metab Bone Dis Rel Res 1982;4:85-93. 31 Lips P, Netelenbos JC, Jongen MJM, Van Ginkel FC, AIthuis AL, Van Schaik CL, Van der Vijgh WJF, Vermiden JPW, Van der Meer C. Histomorphometric profile and vitamin D status in patients with femoral neck fracture. Metab Bone Dis Rel Res 1982;4:85-93. 32 Horsman A, Nordin BEC, Simpson M, Speed R. Cortical and trabecular bone status in elderly WOmen with femoral neck fracture. Clin Orthop 1983;166:143-151. 33 Cummings SR, Are patients with hip fractures more osteoporotic? Am J Med 1985;7;:487-494. 34 Aitken JM. Relevance of osteoporosis in women with fracture of the femoral neck. Br Med J 1984;288:597-601. 35 Wooton R, Reeve J, Slavin G, Tellez-Yudilevich M. Fractured neck of femur in the elderly: ah attempt to identify patients at risk. Clin Sci 1979;57:93-101. 36 Lips AU, Van Ginkel FC, Jongen MJ, Rubertus F, Van der Vijgh WJ, Netelenbos JC. Determinants of vitamin D status in patients with hip fracture and in elderly control subjects. Am J Clin Nutrit 1987;46:1005-1010. 37 Hordon LD, Peacock M. Vitamin D metabolism in women with femoral neck fracture. Bone Mineral 1987;2:413-426. 38 Wiske PJ, Epstein S, Bell NH, Queener SF, Edmondson J, Johnston CC Jr. Increase in immunoreactive parathyroid hormone with age. N Engl J Med 1979;3OO: 1419- 1421. 39 Francis RM, Peacock M, Barkworih SA. Renal impairment and its effects on calcium metabolism in elderly women. Age Ageing 1984;13:14-20. 40 Riggs BL, Jowsey J, Kelly PJ, Hoffman DL, Arnaud CD. Studies on the pathogenesis and treatment in postmenopausal and senile osteoporosis. Clin Endocrinol Metab 1973;2:317-,332. 41 Nilsson BE, Westlin NE. The plasma concentration of alkaline phosphatase, phosphorus and calcium following femoral neck fracture. Acta Orthop Stand 1972;43:SO4-510. 42 Lal SK, Orth MS, Jacob KC, Nagi On, Anna Malai AL, Nair CR. Variations of some plasma components after closed fractures. J Trauma 1976;16:206-211. 43 Hosking DJ. Changes in serum alkaline phosphatase after femoral fractures. J Bone Jt Surg 1978;608:61-65. 44 Weisman J, Salama R, Hare11A, Edelstein S. Serum 24,2S_dihydroxyvitamin D and 25.hydroxyvitamin D concentrations in femoral head fractures. Br Med J 1978;2:1196- 1197. 45 Hordon L, Selby PL, Kruszynska J, Storer J, Taylor GA, Peacock M. Comparison of the biological activity of 25(OH)D, and 24,25(OH),D,. Clin Sci 1984;66:21. 46 Peacock M, Selby PL, Francis RM, Brown WB, Hordon L. Vitamin D deficiency, insufficiency, sufficiency and intoxication. What do they mean? In: Norman AW, et al., eds. Sixth workshop on vitamin D. Berein, New York: Walther de Gruyter, 1985:569-570. 47 Bordier P, Rasmussen H, Marie P, Miravet L, Gueris J, Rykwaert A. Vitamin D metabolites and bone mineralisation in man. J Clin Endocrinol Metab 1978;46:284-294. 48 Rasmussen H, Bordier P. The physiological and cellular basis of metabolic bone disease. Baltimore: Williams and Wilkins Co., 1973:2-63. 49 Villanueva AR, Kujawa M, Mathews CHE, Parfitt AM. Identification of the mineralisation front: comparison of a modified toluidine blue stain with tetracycline fluorescence. Metab Bone Dis Rel Res 1983;5:41-45. 50 Compston JE, Vedi S, Webb A. Relationship between toluidine blue stained calcification fronts and tetracycline IabeIIed surfaces in normal human iliac chest biopsies. Calcif Tissue Int 1985;37:32-35. 51 ArIot M, Edoua-d C, Meunier PJ, Neer RM, Reeve J. Impaired osteoblas! function in osteoporosis: comparison between calcium balance and dynamic histomorphometry. Br Med J 1984;289:517-520. 52 Parfitt AM, Mathew C, Rao D, Frame B, Kleerekoper M, Villanueva AR. Impaired osteoblast function in metabolic bone disease. In: DeLuca HF, Frost HM, Jee WSS, Johnston CC, Parfitt AM, eds. osteoporosis: recent advances in pathogenesis and treatment. Baltimore: University Park Press, 1981;321-330. 53 Davies DF, Shock N. Age changes in glomerular filtration rate, effective renal phSma flow and tubular excretory capacity in adult males. J Clin Invest 1950;29:496-507. 54 Peacock M, Hordon L. Femoral fracture: the role of vitamin D. In: Kleerekoper M, Krane SM. eds. Clinical disorders of bone and mineral metabolism. New York, Basel: Mary Ann Liebert Inc., 1989;26S-271.
ll( 1990)247-259
Elsevier
247
BAM 00320
L.
. Mordon’ and
‘Department of Rheumatology, The General Infirmary, Leeds LSI 3EX, England and ‘General Clinical Research Center, Indiana UniversityHospital, E/534, 926, W. Michigan Street, Indianapolis, IN 46202-5250, USA
(Received 20 December 1989) (Accepted 18 July 1990)
Iliac crest bone histomorphometry, plasma and urine biochemistry and clinical history were examined in 78 unselected patients (68 women, i8 men) at the time of femoral fracture. Histological abnormalities occurred in 56 of the 78 biopsies. The commonest of these was a low bone volume of less than 15% which, irrespective of other abnormal histological features, was present in 37 of the biopsies. On the basis of the histomorphometry, patients could be classified into four main groups. Normal histomorphometry (bone volume >15%, osteoid surfaces 60%) was present in 22 patients, 23 had osteoporosis as the only abnormality (bone volume ~15%. osteoid surface 60%), nine had osteomaiacia (osteoid surfaces >24%, mineralising surface 13 pm) and 13 had decreased mineralising surfaces. Of the remainder, five had increased osteoid surface and six had insufficient osteoid to assess mineralising surface. Plasma and urine biochemistry in the four groups showed that, compared to age-matched controls,, all groups had reduced plasma albumin. In comparison to the group with normal histomorphometry, patients with osteoporosis had a higher plasma calcium (P < O.OI), tubular reabsorption of calcium (P < 0.05) and plasma vitamin D binding protein (P < 0.01); patients with ostewmalacia had a higher plasma creatinine (P < 0.02) and parathyroid hormone (P < 0.02) and lower plasma 24.25dihydroxyvitamin D (P < 0.02), urinary calcium/ creatinine ratio (P < 0.02) and tubular reabsorption of phosphate (P < 0.02). The biochemistry in patients with decreased mineralising surface was no different from patients with a normal biopsy. The prevalence of both osteoporosis and osteomalacia increased with age and, in subjects over the age of 90, osteoporosis occurred in 71% of patients and osteomalacia occurred in 29% of patients. The osteomalacic group were significantly older than the other three groups (P C 0.05). The histomorphometry did not relate to the site of fracture (subcapital or intertrochanteric). A history of stroke, gastrectomy, rheumatoid arthritis, steroid treatment, thyroid disease, alcohol abuse and anticonvulsant therapy was present in patients with femoral fracture but did not relate to any particular histomorphometric classification.
Key words: Osteomalacia; Osteoporosis; Bone histomorphometry; Femoral neck fracture; 1,25(OH),D
Correspondence to: Munro Peacock, M.D. General Clinical Research Center, Indiana University Hospital, N-534,926 W. Michigan Street, Indianapolis, IN 46202-5250,USA. 0169~600!?/90/$03.50@1990Elsevier Science Publishers B.V. (Biomedical Division)
248 Introduction
Femoral neck fracture is the commonest fracture in people over the age of 65 [l] causing considerable mortality and morbidity [2]. In populations of Northern European descent, femoral neck fracture is mainly a disease of elderly women [3-61. Moreover, the incidence of this fracture is increasing over and above the increase in the elderly population, especially in women over the age of 75, the reason for which is not clear. Although many studies have looked at the roles played by osteomalacia and osteoporosis in the pathogenesis of this fracture, their prevalence and relative contributions to the fracture are still disputed. Much of the controversy arises from differences in populations studied, and, particularly in the case of osteomalacia, from different biochemical, radiological and histomorphometric criteria used for diagnosis. The prevalence of osteomalacia in the femoral neck fracture population is of more than academic interest. Whilst advances are being made in the prevention of osteoporosis [7], treatment of established osteoporosis remains difficult [8]. On the other hand, osteomalacia, if detected, can usually be prevented and treated. The aims of our study were to examine bone histomorphometry and its relation to plasma biochemistry and vitamin D metabolites in the femoral neck fracture population to determine whether any clinical or biochemical criteria could. at the time of fracture, identify patients with histologica abnormalities, particularly those of osteomalacia.
Materials and Methods
Two hospitals in Leeds receive unselectively all patients who sustain a femoral fracture. Femoral fracture patients are admitted in rotation by four orthopedic teams and all patients who were treated surgically by one of the four teams were considered for study. Sixty-eight women, aged 52-99 (mean 78) years, and 10 men, aged 45-92 (mean 77) years, admitted to one of the hospitals with femoral neck fracture from March 1984 to February 1986, were studied. During this period, a total of 419 patients (329 women and 90 men) were admitted with femoral neck fracture and 292 of these subsequently received an operation. Our sample is, therefore, unselected and represents 19% of the fracture population admitted and 27% of those operated on for this condition. All patients were of Caucasian origin. Patients were recruited evenly over a 2-year period to exclude any seasonal variation. Patients with subtrochanteric fractures and those known to have malignant disease were excluded from the study. Fracture patients were classified radiologically into two groups, those with subcapital and transcervical fractures, and those with basal cervical and intertrochanteric fractures. The biopsy was performed at the time of surgery which was performed a median of 1 day after fracture (range O-12 days). Informed consent was obtained from all subjects.
249 Methods
Transilial bone biopsies were obtained 1 inch below and behind the anterior superior iliac spine using an 8 mm Bordier trephine. The bone biopsies were fixed in 10% phosphate-buffered formalin for 48 h, dehydrated through graded alcohols, cleared in xylene, embedded in methylacrylate and left to polymerise until hard enough for sectioning. Between 30 and 48 longitudinal sections IO-pm thick were cut from each biopsy using an LKB 2258 PMV heavy duty microtome. Two-thirds of the sections were stained using Goldner to demonstrate osteoid surfaces and one-third in toluidine blue to demonstrate mineralising surfaces. The following measurements were performed on cancellous bone. Bone volume (SWTV) and osteoid volume (OVln/)
Bone volume and osteoid volume were expressed as a percentage of the total tissue volume. The amount of cancellous bone per unit area was determined on 64 fields, four fields on each of 16 sections. Each field was examined using a 25point Zeiss integrating eyepiece and a magnification of ~50 and a point on the grid coinciding with a trabecula was scored as a hit. The number of hits in the four fields of each section was taken as a measure of the percentages of bone in that section. The mean value of the 16 sections was expressed as a percentage volume of bony tissue, assuming the biopsy to be homogenous. In calculating osteoid volume, the number of hits in the four fields of each section coinciding with an ostcoid seam was taken as a measure of the percentage of osteoid in that section. The normal range of BV/TV was defined as greater than 15% [9,10] and that of OV/TV less than 1%. Osteoid surface (OSJBS) and eroded surface (ESIBS)
Osteoid and eroded surfaces were expressed as a percentage of total bone surface. To assess the surface extent of osteoid and eroded surface, a line sampling technique was used em loying the same integrating eyepiece and number of fields and sections, at a magnification of x80. All intersections between the grid lines and trabecular surfaces were counted in each field and the proportion of these intersections coinciding with osteoid or eroded surfaces counted. The results were expressed as a percentage of trabecular surface covered in osteoid or eroded and the normal range for OWBS was taken as less than 24% and for ES/BS 15
12.5 + 1.5
20.0 11.1 19.4 14.6 21.3
BVm + + f f f
0.5 0.02 0.77 0.11 0.33
0.0 cl
0.63 + 0.4
0.25 0.06 2.4 0.20 0.67
OVA’V (%)
(-) 4-13
12.0 z!L0
(-) (-) 20.9 zk 1.4 (-) 12.4 f 1.9
OW @m)
1.1 0.7 2.9 1.0 9.0
8.0 + 1.6 Cl2
f + k f f
0.4 k 0.2 ~24
9.0 8.9 11.9 8.6 23.0
7.5 + 1.5
f 1.3 f 0.7 f 9.7 + 2.7 +. 0.7
ESiBS (%)
36.5 + 6.5
7.7 5.3 53.3 8.7 25.3
OYBS (%)
Results are expressed as means k SEM, the number of men (M) in each group is indicated and variables not measured are shown as (-).
1M 4M 2M
22 23 9 13 3
Normal Osteoporosis Osteomalacia Decreased mineralising surface Increased osteoid surface Increased osteoid surface, decreased mineralising surface, osteoporosis Insufficient osteoid to assess mineralising surface Control range
Sex
N
and age in 78 patients with femoral neck fracture
Classification
Bone histomorphometry
Table 1
+ + + f +
2.4 2.1 7.7 2.6 4.2
(-) >60
35.5 f 5.5
74.9 76.5 36.0 50.6 67.7
MS/OS (%)
252
ranges given [9,10] in Table 1,22 (31%) femoral fracture patients had normal bone histdogy and 23 (32%) had as the only abnormality a bone volume less than 15% and, by definition, were osteoporotic. Osteomalacia, defined as osteoid surface greater than 24%, osteoid width greater than 13 pm and mineralising surface less than 60% [23], was present in nine patients (12%), two of whom also had a bone volume less than 15%. Thirteen patients (18%), had decreased mineralising surfaces, 7 (10%) of whom also had a bone volume less than 15%. Three patients (4%) had increased osteoid surfaces with normal mineralising surfaces, including two with increased osteoid width. Two patients (3%) had increased osteoid surface, decreased mineralising surface but with normal osteoid width and were osteoporotic. In six (8%) of the 78 biopsies there was insufficient osteqid pressnt to assess the mineralising surface. Three of these had bone volume less thzn 1.5t%,and thus of the 78 patients biopsied, 37 (47%) had a bone volume less than 15% and by definition had osteoporosis. Plasma and urine biochemistry in relation to bone histomorphometry (Table 2) For purposes of comparison four groups were defined according to histomorpho-
metric criteria and classified as; normal, osteoporosis, osteomalacia and decreased mineralising surface. The number of subjects in the groups with increased osteoid surface, decreased mineralising surface and normal osteoid width, and increased osteoid surface and normal mineralising surface were too small to analyse statistically. As compared to a group of aged-matched controls, plasma albumin was decreased in all four groups of patients with femoral fracture. Plasma creatinine was also low in all but the osteomalacic r,roup who had a significantly increased concentration (P c 0.001). The vitamin D metabolites were normal in all groups except for the group with decreased mineralisation who had a significant increase in total (P < 0.01) and free (P < 0.05) 1,25(OH),D. Plasma calcium was low in the groups with normal and with decreased mineralisation but parathyroid hormone was in the elderly control range in all groups except the osteomalacics who had a significantly increased concentration (P c 0.001). Plasma phosphate and tubular reabsorption of phosphate was increased in all but the osteomalacic group who had decreased values. As compared to the group with normal histology, patients with osteoporosis had a higher plasma calcium (P < 0.01) plasma DBP (P < O.Ol), and tubular reabsorption of calcium (fp < 0.05). Patients with osteomalacia had higher plasma creatinine (P c 0.02) and PTH (P < 0.02), and lower calciumkreatinine ratio (P < 0.02), TmP (P < 0.02) and plasma 24,25(OH),D (P < 0.02). There were no significant differences in any of the biochemical variables measured between patients with decreased mineralising surfaces alone and those with normal bone histology. Osteoporosis and osteomalacia in relation to age in femoral fracture patients (Fig. I)
The osteomalacic group was significantly older than the other three groups (P < 0.05) (Table 1). Osteomalacia was absent in the 14 patients younger than age 70 but present in 5% of the patients in the 70-79 age group, in 17% in the 80-89 age group
253
Plasma and urine biochemistry
Normal
in patients with femoral neck fracture classified
Osteoporosis
Osteomalacia
Decreased
Elderly
mineralking
controls
surface
Age (years)
79.0+2.2 (II=)‘)
69-93 (n=23)
Plasma CiIlciUm
(mmol/l) Phosphate (mmolit)
1.04+0.0# (11=X) 14.5k-l.tr
AlliitlilW
phosphatasc (LA.
’ 7’+0.02’ _.. _(fit=%)
units)
Crcatinine (qIol/l) Albumin (g/l) ‘(0H)D (nmol/l) 21.2S(OH)2D (nmolil)
1.Z(OH),D (pnK~lll) DBP (frmolil) *Free’
I .25(0H),D (pmal4) PTH
74.1-1-3.8”
97kh
01=22) 3x+ 1AI’ (11= 19) 12.9+1.3 (II- ii) 0.77z!5). I1 (If= 10) 114.3+ I-t.8 (fr= 15) fi.xto.2Y (v= 15) 1.23-tt1.17 (?I= 14) .#l7.2+49.x (If = 15)
Urine CaiCr (mol) OHPCr
9.7+o.H
(If= 19)
(mol)
39+0.x 13.5+ 1.2
93.x+5.8
1.OS~II.OX
0. 14+u.osJ
0.46~0
(tt=7)
(t1=9)
0.029+,().012 (tt
‘7)
13
0.03 1f0.004
o.u+o.
10
0.022+0.003
(a =9)
TmCa
1.Y6+0.Oh
1.83+o.M
1.92+0,004
TmP
(rF=7) 0.74+0. lo”~C
(a=9) 1. itp-I_0.05’
0.93+0.03
(n=7)
(n=9)
Results are expressed as means f. SEM. Significance between rhe group with normal hislolopy and other groups are shown as: “r” < 0.115. “p c 0.02. “P)< 0.01 and between groups and tlderly controls (371 as: dP < 0.05. ‘P 4 0.~11.‘P c 0.001.
254
Fig. 1. Bone volume in relation
to age in patients with femoral neck fracture. Patients with (0) ad ) osteomalaciaare shown.
and in 29% in the 90-99 age group. Osteoporosis was present in 43% of the patients younger than age 70, in 52% in the 70-79 age group, in 53% in the 80-89 age group and in 71% in patients over the age of 90. Ten patients were male and two of these (20%) had osteomalacia. Mean bone volume in the 61 women without osteomalacia was 15.8 + 0.8% (SE) and in the eight men without osteomalacia was 15.6 f 2.1% (SE). Bone histomorphometry in relation to type of femoral neck fracture
Of 78 femoral neck fracture patients studied, 44 (56%) had subcapital fractures and 34 (44%) had intertrochanteric fracture. There was no relationship between bone histomorphometry and the type of fracture. Previous fractures in femoral fracture patients in relation to bone histomorphometry
None of the 10 male patients had previously suffered a femoral neck or wrist fracture. Of the 68 female patients, 15 (22%) had had a previous femoral neck fracture and 13 (19%) had previously sustained a unilateral or bilateral wrist fracture. Four of the seven (57%) female patients with osteomalacia had previously fractured a femoral neck and three (43%) had previously had !i wrist fracture. Although female osteomalacic patients appeared more likely to have had a previous femoral fracture than patients with other histological diagnosis, this trend was not significant (2 with Yates correction = 3.54). Clinical history in femoral neck fracture
A history of stroke (four patients), gastrectomy (five patients), rheumatoid arthritis (six patients), steroid treatment (six patients), thyroid disease (six patients), alcohol abuse (six patients) and anticonvulsant therapy (five patients), was obtained but did not relate to the histological classification.
255
Our study shows that patients biopsied at the time of femoral fracture, thus reducing immobilisation effects, frequently have histomorphometric abnormaiities in cancellous bone. In only 31% of the patients was the biopsy normal. The most frequent abnormality was a bone volume less than 15%. In 32% of patients this occurred in the absence of any other abnormality but in the group as a whole, irrespective of other abnormalities, it occurred in 47% of patients. Osteomalacia, a failure of osteoid to calcify as it is laid down, requires double tetracycline labelling for diagnosis which is difficult to do in patients biopsied at the time of femoral fracture. However, using the coexistence of increased osteoid surface, increased osteoid width and decreased mineralising surfaces, generally accepted criteria indicating the presence of osteomalacia, we found that osteomalacia has an overall prevalence of 12%. Thus it remains a significant abnormality in patients with femoral neck fracture in Britain, and the same is probably true of Europe [24] and Elorth America [25,26]. The occu~ence of both osteomalacia and osteoporosis in our study are age-related, with osteomalacia being particularly high in those over the age of 90 years. The prevalence of osteomalacia in our study was lower than the 20% reported in a previous study from the same center [9]. In that study, however, the criteria for osteomalacia did not inciude osteoid width. Using the same criteria as in that study, the prevalence of osteomalacia in our series would increase to 15%. On the ot igher than that of 2% in a recent study from a similar geographical location to our own [27]. In the later study, osteomalacia was defined as the presence of an osteoid area greater than 5%) making comparison difficult with our study. Five patients (6%) had increased osteoid surfaces, but did not fulfill all the criteria for osteomalacia. The reason for this osteoid increase is not clear. There was no biochemical evidence of primary hyperparathyroidism or clinical history of thyroid disease. Early vitamin D depletion has been shown to cause accelerated bone remodelling and increase osteoid surfaces [28] and this is a possible explanation, particularly in the two very elderly patients who also had low mineralising surfaces and in the one patient on anticonvulsant therapy. The mean BWTV of 15.8 + 5.8 (SD) in female femoral neck fracture patients without osteomalacia was lower than the mean BWTV of 19.6 f 5.6 (SD) reported for healthy American women aged 65-74 [29], and that of 19.8 + 3.9 (SD) described for healthy British women aged 61-80 [30], but there was considerable overlap as has been found in other histomorphometric [9,31] and radiographic studies [32,33]. There were no differences in bone histomorphometry between subcapital and intertrochanteric fracttircr : as found in a previous study [9]. This finding contrasts with others which found that intertrochanteric fractures were present more often in severely osteoporotic women [X,34], and subcapital fractures predominated in osteomaiacic patients [27]. As a group the most striking biochemical abnormality in patients with femoral fracture was 5 subnormal plasma albumin. Low plasma albumin has been noted in
256 other studies [35-373 and is generally assumed to be due to poor nutrition and intercurrent illness. We think it is also related to the trauma and the surgery since 6 months after treatment the low plasma albumin reverts into the normal range [37]. As compared to young normals, increased levels of plasma PTH are a frequent finding in the elderly with [37-391 and without femoral fracture [40]. Multiple factors are probably responsible including renal failure, vitamin D deficiency and insufficiency, calcium malabsorption and poor calcium intake. In our fracture patients the plasma PTH was increased above the young normal range of 125-375 pg/ml but only in the osteomalacic group was it significantly increased from the elderly controls and it was not associated with an increase in erosive surfaces. After fracture, plasma biochemistry changes with an immediate fall in plasma calcium and albumin, and rise in plasma phosphate [41,42], and a late rise in alkaline phosphatase in the second week [43]. Plasma 1,25(OH),D metabolism is also affected by both fracture and hip surgery [37] with a failure of plasma levels to change in response to changes in plasma 25(OH)D. These changes confound the biochemical diagnosis of metabolic bone disease particularly that of osteomalacia. Although the osteomalacic patients in our series had the lowest plasma 25(OH)D levels, they were not significantly lower than those with normal bone histomorphometry. However, 24,25(OH),D was significantly reduced suggesting that the osteomalacic group had the lowest vitamin D status. It has been suggested that 24,25(OH),D plays a specific role in the mineralisation of bone and may be important in femoral fracture [44]. Others, however, have shown that 24,25(OH),D administration has no unique effect on calcium metabolism in osteomalacia [45] and that complete healing of osteomalacia occurs with 1,25(OH),D, administration in the absence of measurable levels of 24,25(OH),D and 25(OH)D [12]. The osteomalacic patients had significantly higher plasma PTH levels and alkaline phosphatase activity, lower TmP, and urine calcium indicating that in this group the overall vitamin D status had progressed from a state of insufficiency to one of true deficiency [46]. It is likely, therefore, that the low 24,25(OH),D simply represents a depression of the 24-hydroxylase by the raised PTH levels. Overali, the biochemistry is helpful in separating the osteomalacics as a group, but less so in the individual. Patients with decreased mineralising surface alone, either with or without osteoporosis, showed no difference in plasma or urine biochemistry from the patients with normal histomorphometry. The mineralising defect may represent an early stage of osteomalacia [47] as this is the first abnormality to occur in the absence of dynamic measurements using tetracycline labelling. However, this appe;irs unlikely to US since the biochemistry of this group is not different from the normal group. The mineralising surface taking up toluidine blue has been shown by some authors to correspond closely to the fraction of osteoid showing tetracycline labelling 148,491although this is disputed [50]. A decrease in the fraction of osteoid showing tetracycline labelling has been reported in patients with osteoporosis [51,52] and it is perhaps significant that over half of our patients who showed decreased mineralising surface but no change in osteoid also had osteoporosis. The decrease in mineralising surface, therefore, may be more a feature of the osteoporotic than the os-
257 teomalacia process. The main clinical condition contributing to osteoporosis and, particularly, to OSteomalacia in femoral neck fracture patients was age. In addition, over half of WOmen with osteomalacia had a previous femoral fracture, compared with a fifth of women with other histology. This may reflect the poor muscle strength and decreased cortical bone [26] of osteomalacia, together with the tendency of Looser’s zones to occur bilaterally. On the other hand, femoral fracture impairs mobility which may precipitate osteomalacia by reducing sunlight exposure. It appears equally likely, therefore, that femoral fracture itself is a risk factor for osteomalacia. It is well known that renal function declines with age [53] and previous work has shown that elderly women with a plasma creatinine over 100,~molll have a GFR of ~50 mllmin, reduced calcium absorption and decreased plasma 1,25(0 ]39]* It is not surprising, tfierefore, that the group with osteomalacia showed the greatest impairment of renal function. surprising is that despite t ey were still able to maintain plasma 1,25( levels in the normal range, albeit with evidence of secondary hyperparathyroidism. The results of this study in femoral neck fracture patients emphasise the high prevalence of abnormalities in bone histomorphometry, the commonest of which is osteoporosis, Osteomalacia is also a significant problem and in the individual patient difficult to diagnose biochemically at the time of fracture. The prevalence of botil osteoporosis and articularly osteomalacia increase with age suggesting that patients with femoral neck fracture as a group have vitamin D insufficiency and calcium malabsorption, which in older patients readily progresses into one of true deficiency resulting in a failure of the osleoid to calcify [54].
We wish to gratefully acknowledge the assistance of the orthopedic surgeons, M.J. Abberton, Mr E.B. Longton, Mr M.A. Nelson and Mr F.F. Silk and their staff, particularly Mr Austen Wynne under whose care the patients were admitted and their bone biopsied. We are also grateful to Mr G.A. Taylor, Dr J. Storer and Dr R.W. Marshall for the biochemical measurements, Dr David Marshall for advice on the statistics Dr J. Aaron and Mr J. Thompson for advice with the histological techniques, and Mrs M. Cromer and Mrs D. Holdsworth for typing the manuscript.
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