Giant Osteoclast Formation and Long-Term Oral Bisphosphonate Therapy

Bisphosphonates are used worldwide to prevent fractures in patients with osteoporosis.1-6 Treatment with these drugs decreases the rate of bone resorption and levels of biochemical markers of bone turnover and causes progressive increases in bone mineral density. The clinical efficacy of nitrogen-containing bisphosphonates is widely believed to result from their potent ability to decrease the number of osteoclasts by promoting their apoptosis.7-9 Once osteoclasts become apoptotic, they are usually quickly ingested by bone marrow phagocytes.10 However, enumeration of osteoclasts in specimens of cancellous bone (bone composed of multiple trabecular structures) obtained from patients treated with nitrogen-containing bisphosphonates shows surprisingly little, if any, change in the number of osteoclasts.11,12 This observation suggests that the mechanism by which these drugs work in vivo may differ from the current thinking. Heretofore, the discrepancy between the antifracture efficacy of bisphosphonates and the absence of any effect on the number of osteoclasts has been attributed to the imprecision of histomorphometric indexes of bone resorption.11 Indeed, osteoclasts in normal persons occupy less than 1% of the cancellous perimeter, so that a decrease in their number might be difficult to detect.13 Alternative explanations for this discrepancy include a greater effect of the drugs on cortical than on cancellous bone, a bisphosphonate-induced decrease in the rate of bone resorption by osteoclasts, and nonadherence to long-term drug therapy.11,12,14

Bisphosphonate-induced inhibition of bone resorption in vitro does not require apoptosis of osteoclasts.15 In addition, administration of bisphosphonates to beagles results in a higher rather than a lower number of osteoclasts owing to prolongation of the osteoclast lifespan.16 These observations suggested to us that nitrogen-containing bisphosphonates may inhibit bone resorption without decreasing the number of osteoclasts and may even increase the number of these cells in cancellous bone. To reconcile the paradoxical findings of these studies, we conducted an in-depth examination of osteoclast morphologic features in bone-biopsy specimens from women who had participated in a trial of alendronate for the prevention of postmenopausal osteoporosis.3
Methods
Study Population and Design

Transilial bone-biopsy specimens were available from the trial by McClung et al., which was a 3-year (1994 to 1997), double-blind, randomized, placebo-controlled, dose-ranging trial of alendronate in 447 healthy postmenopausal women 40 through 59 years of age who had entered menopause 6 to 36 months before enrollment.3 The trial by McClung et al. was a prevention trial, and the bone mineral density of the patients was within 2 SD above or below normal peak adult values at the initiation of the trial. However, the bone mineral density of the patients at baseline was approximately 10% below the mean value for young adult women, and the bone mineral density in patients receiving placebo decreased by 3 to 4% at the spine, femoral neck, and trochanter during the trial. This result indicates that these women were having postmenopausal bone loss.3 Women who had disorders of bone and mineral metabolism, smoked more than 20 cigarettes per day, or consumed three or more alcoholic drinks per day were excluded. The patients were randomly assigned to one of five groups: those receiving placebo for 3 years; oral alendronate at a dose of 1, 5, or 10 mg per day for 3 years; or oral alendronate at a dose of 20 mg per day for 2 years, followed by placebo for 1 year. The original trial had shown that alendronate at a dose of 5 or 10 mg per day for 3 years or at a dose of 20 mg per day for 2 years, followed by placebo for 1 year, increased total body bone mineral density and bone mineral density at the lumbar spine, femoral neck, and trochanter by 1 to 4% from baseline. Patients receiving alendronate at a dose of 1 mg per day had a lower rate of loss of bone mineral density than those receiving placebo. Alendronate also decreased the levels of biochemical markers of bone turnover as compared with baseline.1-3

After 3 years of treatment, 55 patients volunteered to undergo a transiliac bone biopsy to determine whether the drug had impaired skeletal mineralization, as had occurred with etidronate, a first-generation bisphosphonate.17 To label the bone for the measurement of the mineral appositional rate and the mineralizing perimeter, the patients were given oral tetracycline hydrochloride (250 mg four times per day) 19, 18, 7, and 6 days before the biopsy. Thus, each patient underwent two periods of tetracycline labeling separated by an interval of approximately 2 weeks. Fifty-five biopsy specimens 7 mm in diameter were obtained; 51 of these included enough of the two cortexes and the intervening cancellous bone to permit histomorphometric evaluation. All 15 centers that contributed specimens received approval from their institutional review boards, and all patients provided written informed consent. Additional approval for reexamining the specimens was obtained from the review board of the University of Arkansas for Medical Sciences, where the specimens were held and read centrally. Each specimen was coded so that the reader was unaware of the patient's study-group assignment. The finding of normal osteoid thickness and a decreased mineralizing surface (the length of double-tetracycline-labeled cancellous bone plus half the length of single-tetracycline-labeled cancellous bone) in the alendronate groups as compared with the placebo group has already been reported.3 To our knowledge, the rates of bone formation and mineral apposition and the number of osteoclasts have not been previously reported.
Histomorphometric Measurements

Within 24 hours after the biopsy, the specimens were fixed in iced Millonig's phosphate-buffered 10% formalin at pH 7.4 and sent by express mail to the University of Arkansas for Medical Sciences. At the university, the specimens were dehydrated in graded ethanol solutions and embedded undecalcified in methylmethacrylate.18 The tungsten–carbide D-profile rotary microtome blades (Delaware Diamond Knives) were sharpened every 2 weeks to maintain the quality of the sections. Longitudinal sections, 5 μm in thickness, were taken from one third and one half the depth of the specimens. Two sections from each depth were left unstained for examination of the tetracycline labels, and two sections were stained by a modification of the Masson technique to enhance nuclear detail.13 Additional sections were stained for tartrate-resistant acid phosphatase (TRAP).19,20 TRAP staining was performed to confirm that the abnormal cells were indeed osteoclasts, but TRAP staining impairs nuclear detail. Therefore, all osteoclast measurements were performed on the Masson-stained sections. The coverslips were compressed with 2-oz (approximately 60-g) fishing weights for 30 minutes to facilitate the production of smooth, planar sections free of distortion.

The histomorphometric examination was performed with the use of a digitizer tablet (OsteoMetrics) interfaced to a Zeiss Axioscope with a drawing-tube attachment, as previously described.13,17,18,21 The total number of osteoclasts on or adjacent to cancellous bone was expressed as the number per millimeter of cancellous bone perimeter. A giant osteoclast was defined as one having at least three of the following characteristics: more than eight nuclear profiles (two-dimensional images of a section taken through three-dimensional objects), detachment from bone, a shallow or absent resorption cavity, pyknotic nuclei, or the presence of other mononuclear cells interposed between the bone perimeter and the osteoclast. The osteoclasts of patients in the placebo group had fewer than three of these characteristics, but they infrequently showed some small degree of detachment or nuclear pyknosis. The osteoclasts were then further classified according to the number of nuclear profiles that could be counted in the 5-μm-thick sections; these sections contained only a small portion of the giant osteoclasts, which were 80 μm to more than 100 μm in thickness. A normal osteoclast is shown in Figure 1Figure 1Normal Multinucleated Osteoclast Tightly Adherent to Bone from a Patient Receiving Placebo..
Measurement of Apoptosis in Undecalcified Bone Sections

Apoptosis of osteoclasts was detected in additional sections taken from the blocks by in situ end labeling with the use of the Klenow FragEL DNA Fragmentation Detection Kit (Oncogene Research Products), as previously described.22 Osteoclasts positive for in situ end labeling also had morphologic changes that included discretely condensed chromatin, nuclear fragmentation, nuclear peripheral beading, and cell shrinkage. To be counted as apoptotic, an osteoclast had to be stained by in situ end labeling and have at least three of the four morphologic criteria. For negative controls, we used archival specimens from patients with renal osteodystrophy who had abundant osteoclasts due to secondary hyperparathyroidism.18
Statistical Analysis

To evaluate group changes in the histomorphometric data, we used one-way analysis of variance, followed by Dunnett's multiple-comparison test.23,24 The data are reported as means and standard errors, unless otherwise indicated; P values of less than 0.05 were considered to indicate statistical significance. The total oral dose of alendronate was calculated by multiplying the daily dose by the number of days it was taken. Comparisons of interest were specified a priori. Consequently, when comparing groups with respect to the number of normal-appearing osteoclasts, we tested only the hypothesis that the number of such osteoclasts was higher in the group receiving 10 mg of alendronate per day (a commonly used dose) than in the placebo group. Pearson correlation coefficients were used to test for an association between the number of osteoclasts and the total amount of alendronate administered. Patients for whom treatment with alendronate was stopped after 2 years were excluded, although similar results were obtained when all patients were included. All cell counts, including zeroes, were included in the analyses. The residuals from a linear regression of the total number of osteoclasts against the total amount of alendronate administered passed the test of normality; this result supports the use of Pearson correlation coefficients, although similar results were obtained with the use of Spearman coefficients.
Results
Standard Histomorphometric Findings

To establish that alendronate reduced bone turnover, as expected, we examined histomorphometric measurements of bone formation (Table 1Table 1Bone Histomorphometric Features after Long-Term Treatment with Alendronate.). The rate of bone formation was diminished to the same extent in the groups receiving 1, 5, or 10 mg of alendronate per day (P<0.003 r="0.50," r="0.55," r="0.40,">

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