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Division of Endocrinology/Metabolism, the Center for Osteoporosis and Metabolic Bone Diseases and the McClellan Veterans Affairs Medical Center Geriatric Research, Education, and Clinical Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Address all correspondence and requests for reprints to: Robert S. Weinstein, M.D., Division of Endocrinology and Metabolism, Slot 587, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, Arkansas 72205. E-mail: rweinstein{at}medlan.uams.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Loss of gonadal function in either sex increases osteoclast precursor formation in the bone marrow, leading to an increase in the number of osteoclasts on cancellous bone, thereby resulting in increased bone resorption and loss of bone; sex steroid replacement prevents these changes (2, 3). Intriguingly, besides the up-regulation of osteoclastogenesis after loss of sex steroids, there is an increase in the production of osteoblast precursors in the bone marrow. Therefore, changes in the development of bone cell precursors in the marrow can explain not only the increase in bone resorption that follows loss of sex steroids, but also the expected and well established increase in the rate of bone formation (1). It has, however, remained unclear whether the increased bone resorption caused by gonadal steroid deficiency is mediated by releasing a direct restraining effect on osteoclasts or indirectly, via the bone marrow stromal/osteoblastic cells that support osteoclastogenesis.
A mechanism to account for the potent effects of sex steroids on osteoclastogenesis has been provided by the evidence that both estrogens and androgens act through receptors expressed in bone marrow stromal/osteoblastic cells to down-regulate the production of interleukin-6 (IL-6), an osteoclastogenic cytokine that is produced in the bone microenvironment, predominantly by cells of the stromal/osteoblastic lineage. In addition, sex steroids decrease the expression of the ligand-binding subunit of the receptor for IL-6 and the signal-transducing subunit of the receptor for IL-6 and related cytokines (3, 4, 5, 6). Consistent with this evidence, sex steroid deficiency increases the production of IL-6 as well as the expression of the IL-6 receptor (7, 8, 9). In support of the role of stromal/osteoblastic cell-derived local mediators in the pathophysiology of the bone loss associated with gonadal deficiency, increased production of IL-6 along with an increase in the expression of the receptor for this cytokine have also been implicated in the pathophysiology of seven more disease states characterized by increased bone resorption: Paget’s disease, multiple myeloma, hyperparathyroidism, hyperthyroidism, McCune-Albright syndrome, Gorham-Stout disease, and rheumatoid arthritis (6).
Senescence-accelerated mice (SAMP6) exhibit early osteoporosis and spontaneous tibial fractures while maintaining intact reproductive function. SAMP6 and the control strain SAMR1 are substrains of AKR/J developed by brother-sister mating. They have been maintained as separate substrains for over 50 generations (10). The calcium, phosphorus, and hydroxyproline contents per dry wt of bone are indistinguishable between the two substrains (11). At 3 weeks of age, the bone mineral densities (BMDs) at the global (total body BMD minus the head), spine, and hindquarters regions of interest of SAMP6 and SAMR1 are similar (12). The osteopenic phenotype of SAMP6 compared with that of SAMR1 becomes manifest at the spine in 3- to 4-month-old adults and at the global, spine, and hindquarters regions of interest in 15- to 16-month-old animals. The BMD in the 15- to 16-month-old SAMR1, however, is not significantly different from that found in the 3- to 4-month-old SAMR1 mice. This accelerated osteopenia is inherited as a polygenic quantitative trait, as demonstrated by the unimodal distribution of the BMD, with greater variance in F2 mice than in F1 hybrids or the parental strains (13). Using polymorphic microsatellite markers, SAMP6 mice differ from SAMR1 mice at 39% of their 20 chromosomes (14). Thus, SAMR1 is the best available control for SAMP6.
We recently reported that these mice develop normally, and at 3 weeks of age they show similar production of osteoblast progenitors capable of forming mineralized colonies in ex vivo marrow cell cultures (CFU-OB) and similar rates of bone formation compared with SAMR1. At 3 months of age, however, female SAMP6 mice have a 3-fold decrease in the production of CFU-OB, and only 1/10th of the bone formation rate compared with SAMR1. Moreover, in this model of defective osteoblastogenesis, the expected increase in osteoclast development in the bone marrow after gonadectomy is blunted (12). That the defect in the development of osteoclasts is secondary to a decrease in the number of stromal/osteoblastic cells rather than to inadequate numbers of osteoclast progenitors is indicated by the restoration of osteoclast formation in SAMP6, when marrow cells from these animals are cocultured with stromal/osteoblastic cells from normal mice. Furthermore, the numbers of granulocyte-macrophage colony-forming units, the presumed osteoclast precursors (15), are identical in SAMR1 and SAMP6 (12).
In view of the above, SAMP6 mice provide a unique opportunity to investigate whether the adverse effects of sex steroid deficiency on the skeleton are mediated by the release of a direct restraining effect on osteoclasts and are, therefore, independent of the concurrent up-regulation of osteoblast development, or whether they are linked to the increased osteoblastogenesis. Because the antiosteoporotic effects of estrogen and androgen seem to be exerted by similar mechanisms, and after ovariectomy, alterations in bone growth and body weight can be confounding factors when attempting to attribute skeletal differences solely to loss of sex steroids (16, 17, 18, 19, 20), we studied the effects of orchidectomy, rather than ovariectomy, to avoid these potential difficulties. The results of our studies demonstrate that the expected changes in bone cell progenitors, histomorphometry, and BMD after orchidectomy either were absent or greatly attenuated in SAMP6 mice. This evidence suggests that the adverse effects of sex steroid withdrawal on bone are, indeed, mediated via cells of the bone marrow stromal/osteoblastic lineage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BMD determinations were made before surgery on day -1 and on the last day of the experiment, day 21 (12). Tetracycline HCl (30 mg/kg BW) was given ip 15 and 20 days after the operation. On day 21, bone marrow aspirates were obtained from the right femur for ex vivo marrow cell cultures, and the cancellous bone of the left distal femoral secondary spongiosa and lumbar vertebrae were prepared for histomorphometric analysis.
Bone densitometry
Dual energy x-ray absorptiometry was used to determine the BMD
of the animals used in this project (Fig. 1
). We adapted
this useful tool for the study of sequential measurements in live mice
by making hardware and software changes to a clinical bone densitometer
as previously described (12). All scans were performed with the animals
positioned prone and spread with tape attached to each limb on a
precision-milled acrylic block. The acrylic softened the photons from
the 140/70 peak kilovolts (kVp) x-rays that were generated by the
QDR-2000 Plus densitometer (Hologic, Waltham, MA) and thus allowed
better visual resolution of the murine skeleton on the digital
radiograph (Fig. 1). Nonlinear beam-hardening effects were also
minimized by use of the acrylic block.
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Bone histomorphometry: fixation, embedding, sectioning, and
staining
The distal femur and lumbar vertebrae were fixed in 4 C
Millonig’s phosphate-buffered 10% formalin, pH 7.4, as previously
described (12, 21, 22). The methyl methacrylate blocks were ground on a
water-cooled grinder (Torit Corp., St. Paul, MN) to just larger than
the size of the specimen. Sectioning was assisted by a stereomicroscope
mounted on the microtome (HM350, Carl Zeiss, Inc., Thornwood, NY) and
focused on the cutting plane. Murine osteoclast recognition was
enhanced by staining for tartrate-resistant acid phosphatase (TRAPase)
on 3-µm thick longitudinal sections counterstained with 1.0%
toluidine blue (pH 2.8). Positive control sections of morphologically
recognizable osteoclasts from a patient with severe renal
osteodystrophy (23) were included in each TRAPase staining procedure.
Adjacent 7-µm thick sections were left unstained for
epifluorescence.
Histomorphometric analysis
The histomorphometric examination was performed with a computer
and digitizer tablet (version 3.00, OsteoMetrics, Atlanta, GA)
interfaced to a Zeiss Axioscope with a drawing tube attachment as
previously described (12, 24, 25, 26, 27). All measurements were
two-dimensional, confined to the secondary spongiosa, and made at x400
magnification (numerical aperture, 0.75). The terminology used is that
recommended by the histomorphometry nomenclature committee of the
American Society for Bone and Mineral Research (28).
Static measurements of cancellous bone
The cancellous bone area was expressed as the percentage of
tissue area, including bone and marrow. The trabecular width and
spacing (the distance between the midpoints of adjacent trabeculae) and
wall width (the distance from a cement line to the quiescent perimeter;
Fig. 2, A and B) were measured directly (25, 26). The
osteoblast perimeter was expressed as the number of osteoblasts
palisading osteoid per mm cancellous bone perimeter. The osteoclast
perimeter was expressed as a percentage of the total cancellous
perimeter covered by TRAPase-positive osteoclasts and as the number of
osteoclasts per mm cancellous bone perimeter.
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Detection and quantification of osteoblast progenitors
Marrow cells were obtained by excising the ends of a
disarticulated femur and flushing the marrow with 5 ml phenol red-free
MEM (Life Technologies, Gaithersburg, MD) containing 10% FBS (HyClone,
Logan UT), using a syringe and a 25-gauge needle. After the aspirate
was rinsed and resuspended using a 23-gauge needle to obtain a single
cell suspension, the nucleated cell count was determined using a
Coulter counter (Coulter Electronics, Hialeah, FL). Cells were seeded
at 1.5 x 106 or 2.5 x 106
cells/10-cm2 well for the determination of CFU-F and
CFU-OB, respectively, and maintained in phenol red-free MEM containing
15% preselected FBS, 50 µM ascorbic acid, and 10
mM ß-glycerophosphate; half of the medium was replaced
every 5 days. For the determination of CFU-F, cells were cultured for
10 days and then stained for alkaline phosphatase and counterstained
with hematoxylin. Colonies of cells containing a minimum of 20 cells
were designated CFU-F and were enumerated without regard to the extent
of alkaline phosphatase staining, which served only as a positive
control for the presence of osteoblastic cells in the cultures. For the
determination of CFU-OB, the cultures were maintained for 25–28 days,
fixed in 50% ethanol and 18% formaldehyde, and then stained for
calcium deposits using 2% alizarin red or Von Kossa’s method. The
colonies exhibiting a nodule of mineralized bone matrix were designated
CFU-OB. In these cultures, the individual colonies containing bone
nodules were well separated (29, 30, 31).
Statistics
To evaluate the development of orchidectomy-induced
osteoporosis, we tested for differences in the serial bone densitometry
values among sham-operated and orchidectomized mice using both the
absolute measurements (milligrams per cm2) and the
sequential percent changes in BMD over the course of the experiment
(16, 32). To evaluate the changes in bone histomorphometry and ex
vivo marrow cell cultures, we used Student’s t test to
assess significant differences between the means of two groups after
testing for equivalence of variances and normal distribution of data.
If the data sets did not have equal variances, Welch’s formula for the
degrees of freedom for the test was used. Data that were not normally
distributed were analyzed using the Mann-Whitney U statistic.
Correlation coefficients were calculated to test for an association
between two independently measured variables. P < 0.05
was considered significant (33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In both
substrains, peak spinal bone mass was reached between 90–150 days of
age. The difference between SAMR1 and SAMP6 is clearly illustrated as
it develops in the adult (91- to 149-day-old) mice: 56.9 ± 3.3
(±SD) vs. 53.1 ± 1.9 mg/cm2
(P < 0.05) (12). Accordingly, we decided to use 4- to
5-month-old animals for the subsequent experiments to avoid the
potentially confounding effects of growth.
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Analysis of bone histomorphometry and BMD
Wall width, an index of the amount of bone made by a previous team
of osteoblasts, was significantly decreased in the cancellous bone of
the femoral secondary spongiosa in sham-operated SAMP6 compared with
that found in SAMR1 [8.9 ± 1.7 (SD) vs.
15.9 ± 0.9 µm; P < 0.001; Table 1]. Furthermore, there was a 16% difference between
the sham-operated SAMP6 and SAMR1 in the final measurements of global
and spinal BMD [44.4 ± 1.8 vs. 53.1 ± 2.1
mg/cm2 (P < 0.002) and 49.3 ± 2.0
vs. 58.9 ± 2.9 mg/cm2 (P
< 0.003), respectively]. Femoral wall width had a direct linear
relationship with the final measurements of global and spinal BMD
(r = 0.98; P < 0.001 and r = 0.97;
P < 0.001, respectively). Concurrent determination of
CFU-F and CFU-OB was performed on marrow cells obtained from these
mice. Direct correlations were found between the femoral cancellous
bone area and both CFU-F (r = 0.82; P < 0.02) and
CFU-OB (r = 0.76; P < 0.05). Thus, compared with
controls, SAMP6 mice exhibited decreased wall width and BMD associated
with diminished osteoblastogenesis.
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and Fig. 4).
Similarly, the rate of bone formation per bone perimeter was increased
(4.5-fold; P < 0.001), as was the rate of bone
formation per bone area or bone turnover (5.5-fold; P
< 0.001) in SAMR1. In SAMP6, however, there were no significant
differences between orchidectomized and sham-operated animals in any of
these determinations.
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), but no significant
changes were observed in SAMP6. The cancellous bone area decreased by
61% (P < 0.001), and the trabecular spacing increased
by 160% (P < 0.01) in orchidectomized SAMR1. Unlike
the previous determinations that show no difference in orchidectomized
vs. sham-operated SAMP6, cancellous bone area and trabecular
spacing were significantly different in orchidectomized vs.
sham-operated SAMP6. Nonetheless, the differences in SAMP6 were
substantially smaller than those found in SAMR1, suggesting that, at
least as far as these two measurements are concerned, orchidectomy had
a small, but substantially blunted, impact in SAMP6.
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). In contrast, BMD did not
significantly change after orchidectomy in SAMP6. Trabecular width did
not change after orchidectomy in either substrain, indicating that the
loss of bone density documented in SAMR1 was due to the removal of
entire trabecular profiles by osteoclasts.
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), whereas no change occurred in
orchidectomized SAMP6. In parallel, there was a significant
augmentation of the rates of mineral appositional and bone formation in
SAMR1, whereas no such changes occurred in SAMP6. Lumbar cancellous
bone area had a direct relationship with both the final global and
spinal BMD (r = 0.68; P < 0.01 and r = 0.66;
P < 0.01, respectively).
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). The change in BMD was also negatively
correlated with osteoclasts per mm (r = -0.59; P
< 0.02) and concurrent determinations of CFU-OB (r = -0.55;
P < 0.02), indicating that low BMD was associated with
both increased numbers of cancellous osteoclasts and osteoblast
progenitors.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have attempted to probe into the mechanism of the deleterious effects of acute androgen deficiency on bone. Specifically, prompted by our earlier findings that loss of sex steroids does not result in the expected up-regulation of osteoclastogenesis in the bone marrow of SAMP6, we wished to determine whether the bone of these mice will be protected from the loss of sex steroids. Our results demonstrate that orchidectomy fails to increase CFU-F and CFU-OB, the presumed osteoblast precursors in the bone marrow of SAMP6. In addition, orchidectomy fails to increase the activation frequency, rates of bone formation, and number of osteoclasts and osteoblasts per cancellous perimeter. Furthermore, orchidectomy does not change BMD and only minimally decreases cancellous bone area in these mice. Based on extensive evidence from our earlier work showing a close relationship between the cellular changes in the bone marrow and on cancellous bone and the bone mass (12), we believe that these findings do not represent isolated observations, but, rather, a continuum from the single cell level to the gross densitometric picture. The detection of some small changes in bone histomorphometry in SAMP6 after orchidectomy supports the concept of a continuum, as osteoblast precursors are diminished, but not completely absent, in these animals.
Bone histomorphometry is a highly discriminating technique for the cancellous changes after orchidectomy (32). In the application of such analysis in our study, we were careful to select an experimental design that would minimize confounding factors. Thus, we performed a large preliminary analysis of the BMD in the mice used in these experiments to restrict our analysis to the loss of sex steroids as opposed to changes due to growth. Our observation that the SAM mice reach peak adult bone mass between 90–150 days of age is consistent with the findings of Matsushita et al. (11) showing that longitudinal bone growth and bone ash in these animals was also maximized at this age. Histomorphometry of the murine skeleton is, however, difficult because of the small amount of cancellous tissue and the reduced size and granularity of the osteoblasts compared with those in rat or human bone. These features may contribute to the high variation in histomorphometric measurements of the osteoblast perimeter. We have previously reported that the technique precision of this measurement in clinical material is 18.8% (39). In mice, the osteoblast perimeter could have been overestimated because of the inclusion of preosteoblasts or osteoblasts soon to become flattened lining cells. Methods to identify matrix-secreting osteoblasts by their expression of specific messenger RNA should allow more precise characterization of these cells (40). Despite these difficulties, the histomorphometric changes clearly show that orchidectomy had a far more pronounced effect on SAMR1 than on SAMP6.
In addition, we chose to study the impact of sex steroid deficiency on bone using male as opposed to female animals because orchidectomy does not cause a change in body weight, whereas ovariectomy does. Moreover, as the histomorphometric studies of remodeling bone were performed in the secondary spongiosa of adult SAMR1 and SAMP6 mice, our findings should be independent of the effects of growth or modeling. The clearly documented cement lines indicate that the histomorphometric examination in this report was restricted to sites undergoing remodeling activity. Cement lines are deposited after resorption is finished and before formation begins and thus demarcate the reversal between removal of the old bone and replacement by the new during turnover.
Our findings demonstrate for the first time that the presence of normal numbers of cells of the osteoblastic lineage is indispensable for the up-regulation of osteoclastogenesis that follows loss of androgens. Furthermore, the increase in the production of both osteoblast and osteoclast precursors in the bone marrow accounts for the increased activation frequency and loss of bone, as none of these changes happen in a model with a primary defect in its ability to produce osteoblast progenitors in the bone marrow. These studies cannot, however, exclude the possibility that androgens may also have some direct effect on osteoclasts. After gonadectomy, loss of bone is partly due to expansion of the remodeling space (41) and to the loss of scaffolding that follows perforation of the cancellous network by osteoclasts (42); it may also involve an inadequate number of osteoblasts available to reconstitute the previously eroded cavities and help maintain bone mass. One possible scenario to account for this is that the impact of sex steroid deficiency on osteoblast progenitors in the bone marrow may cause a relatively larger increase in the cells that support osteoclast development as opposed to those progenitors that become mature bone-synthesizing osteoblasts. Another possibility is that sex steroid deficiency, even though it up-regulates osteoblastogenesis, may result in a decrease in the activity of differentiated osteoblasts (43). Alternatively, sex steroid deficiency, which is known to delay apoptosis of osteoclasts (44), may not have such an antiapoptotic effect on osteoblasts, thereby creating an imbalance in bone turnover. Of course, additional work will be required to test these possibilities.
In conclusion, the results of this study demonstrate that cells of the osteoblastic lineage are essential mediators of the changes in the rate of bone remodeling and the loss of bone mass that occur after the loss of androgens and that these changes are blunted when osteoblast precursors are diminished. In addition, our findings highlight the interrelationships of the progenitor cell determinations, histomorphometric analysis, and BMD measurements and strengthen the evidence for a tight linkage among the development of osteoblast progenitors in the marrow, osteoclastogenesis, and loss of bone mass.
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Acknowledgments |
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Footnotes |
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Received March 14, 1997.
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