To evaluate the ability of estrogen replacement therapy (ERT) to prevent changes in trabecular bone volume (BV/TV) and connectivity beginning either at ovariectomy (OVX) or 5–13 days after OVX in adult female rats, the right proximal tibial was examined by three-dimensional X-ray tomographic microscopy (XTM) in vivo. Animals had XTM scans of the right tibia and then were randomized into six groups (n = 9). Groups 2–6 had bilateral (OVX), while group 1 was sham-ovariectomized (OVXd) on day 0. Animals were treated with vehicle (groups 1 and 2) or 17β-estradiol therapy (ERT) at 10 μ g/kg three times per week starting at days 0, 5, 8, and 13 post-OVX (groups 3, 4, 5, and 6), until day 50 when they were rescanned by XTM and sacrificed. Trabecular bone structural variables were calculated from XTM data (BV/TVx and β1/BV/TVx) and standard histomorphometry. Trabecular bone volume (BV/TVx) and the trabecular connections per cubic millimeter of trabecular bone (β1/BV/TVx) were maintained in both sham-OVXd animals and OVX animals given ERT from the time of OVX. However, OVX + vehicle–treated animals lost 54% BV/TVx and 46% β1/BV/TVx (p < 0.01 from day 0). BV/TVx and β1/BV/TVx decreased rapidly post-OVX to −22% and −25% at day 13 (p < 0.01 from day 0). ERT initiated at day 5, 8, and 13 post-OVX restored BV/TVx to baseline values at day 50 by modestly increasing trabecular plate thickness; however, β1/BV/TVx was reduced in all OVX groups when compared with their baseline values. ERT also caused a significant reduction in bone turnover compared with OVX + vehicle; however, resorption was suppressed more than formation. These results demonstrate that ERT can restore the lost trabecular bone, but not trabecular connectivity, that occurs soon after OVX by allowing bone formation to continue in previously activated bone remodeling units while suppressing the production of new remodeling units. This may be the mechanism by which prompt intervention with estrogen and other antiresorptive agents can restore bone mass that has been lost from the increase in remodeling space, and thereby reduce the risk of osteoporotic fractures in postmenopausal women.
Estrogen deficiency in humans results in both bone loss and architectural deterioration that lead to an increased incidence of osteoporotic fracture.(1,2) Changes in integrated cortical and cancellous bone mass that predict the risk for osteoporotic fractures are readily quantitated in vivo by densitometry.(3) However, serial changes in trabecular bone structure are difficult to measure because safe noninvasive techniques that visualize bone trabeculae repeatedly are not yet widely available.(4,5)
We have developed and validated a three-dimensional (3D) method to image the trabecular lattice in vivo, using high-resolution X-ray tomographic microscopy (XTM).(6,7) XTM-based studies of ovariectomized (OVXd) rats show that trabecular bone connectivity and bone volume deteriorate by 5–8 days post-OVX,(8) a finding similar to other reports in which 2D trabecular structural analysis from rats at 10 days post-OVX.(9) Antiresorptive agents, like estrogen and the bisphosphonates, have been shown to preserve bone mass in acute estrogen deficiency in both clinical and animal studies.(10-21) In most of these animal studies, the antiresorptivity was initiated immediately at the onset of estrogen deficiency, and trabecular bone volume (BV/TV) was maintained or augmented mildly.(12,14,19,21,22) The data from these studies, collected using 2D histomorphometric techniques, suggest that 3D structural analysis of similar animals could provide further insight into the events surrounding acute estrogen depletion bone loss.
Based on the observation that acute estrogen deficiency causes rapid (5–8 days) deterioration in trabecular structure, we hypothesized that estrogen replacement therapy (ERT) begun at the time of ovariectomy (OVX) would maintain trabecular bone connectivity and volume. We further hypothesized that delaying ERT until 5–13 days post-OVX would allow mild deterioration of trabecular bone structure but would permit permanent stabilization of the mildly deteriorated structure.
The purpose of this study was to serially assess the trabecular microarchitecture status in individual OVXd adult female rats that began ERT at 1, 5, 8, and 13 days post-OVX.
MATERIALS AND METHODS
Fifty-four, 1-month-old virgin Sprague-Dawley rats were purchased from Charles River Laboratory (San Jose, CA, U.S.A.) and age matured to 6 months in the Animal Care Facility of the University of California at San Francisco. The animals were housed individually in wire cages, given free access to food (Teklad, Madison, WI, U.S.A.) and tap water, and treated according to the USDA animal care guidelines with the approval of the University of California at San Francisco Committee on Animal Research.
At age 6 months, an XTM scan of the right proximal tibia of all animals was performed. After the scan, animals were randomized by weight into six experimental groups (n = 9). Group 1 was sham-OVXd and groups 2–6 had bilateral OVX performed by the dorsal approach.(23) Animals in groups 1 and 2 were treated with vehicle from day 1 through day 50, 3 days a week; animals in group 3 had 17β-estradiol (ERT) at 10 μg/kg three times per week from day 1 through day 50. XTM scans were repeated on day 5 (group 4), day 8 (group 5), and day 13 (group 6) post-OVX. ERT (10 μg/kg three times per week) was initiated after the second XTM scan in groups 4, 5, and 6 and continued until day 50. At day 50, all animals were rescanned by XTM and sacrificed. Two doses of calcein (Sigma Chemical Company, St. Louis, MO, U.S. A.) at 10 mg/kg of body weight were given 10 days and 3 days before sacrifice.
For ERT, 17β-estradiol (Sigma Chemical Co.) was dissolved in a vehicle of 95% corn oil and 5% benzyl alcohol.(14) For surgery, necropsy, and scanning, the animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride and xylazine at doses of 30 mg/kg and 10 mg/kg of body weight, respectively. Each XTM scan lasted 30–40 minutes. After OVX, the animals were housed three per cage at 25°C with a 13 h light/11 h dark cycle. OVXd animals were fed 15–18 g of food daily, the mean food intake of the sham-OVXd animals. On the day preceding each XTM scan, the rats were housed in individual metabolic cages for collection of a 24-h urine sample.
On day 50, all of the rats were anesthetized. Seven milliliters of blood was obtained from the inferior vena cava, causing death by exsanguination. Success of OVX was confirmed by failure to detect ovarian tissue and by the observation of marked atrophy of the uterine horns. Both legs were disarticulated at the hip. The right femur and tibia were wrapped in saline-soaked gauze and stored at −70°C. The left tibia and femur were cleaned of excess muscle and soft tissue. The anterior eminence of the left tibia was shaved with a razor blade, barely exposing bone marrow, and the femur and tibia were placed in 10% phosphate-buffered formalin for 24 h and transferred to 70% ethanol.
X-ray tomographic microscopy
For each scan, the rat was secured on a rotating stage with the right hindlimb elevated.(6) The radiation source was the University of California National Laboratory's 31-pole X-ray wiggler. The X-ray energy was made nearly monochromic at 25 keV by using a single crystal silicon (220) monochromator with a resolution of 23 μ m/voxel. The right tibia was scanned from the growth plate distally in 23-μ m slices for a total of 60 slices, encompassing the region ∼1.0–2.5 mm distal to the growth cartilage-metaphyseal junction. A region of trabecular bone (secondary spongiosa) from 0.9–1.9 mm distal to the growth cartilage-metaphyseal junction was extracted from each image.(6-8) For each of the scans, care was taken to select exactly the same region by first identifying the anterior eminence of the proximal tibia, then the growth cartilage-metaphyseal junction. During scan analysis, the operator was blinded to treatment and scan order, but not to rat identity.
The volumetric data were thresholded into binary data sets containing only 0′s and 1′s, where the 1′s represented voxels occupied by bone and the 0′s represented voxels occupied by bone marrow. A local threshold algorithm described by Luo et al. was used.(24) Artifacts created during thresholding were removed with a two-step process. In the first step, all bone fragments not continuously connected to the trabecular bone network were removed. In the second step, all artifactual porosity created during thresholding was removed. Though these artifacts accounted for < 0.001% of the total bone volume, their removal was essential for correctly determining the topological connectivity of the trabecular bone.
The thresholded volumetric data were analyzed by two methods, as described previously.(6) The fraction of voxels lying inside the contours per unit section area AA were equated directly to the BV/TV (2D). These two measured values of trabecular bone area (Tb.Ar) and perimeter (Tb.Pm) were then used to calculate the trabecular bone thickness (Tb.Th), trabecular separation (Tb.Sp.), and trabecular number (Tb. N) with the plate model.(25)
Second, cluster analysis was performed on the trabecular bone structures in the 3D images.(26) Cluster analysis identified all the trabecular bone that was continuously interconnected and also identified any isolated structures that were disconnected from the surrounding cortical bone and trabecular structure. Cluster analysis, therefore, provided a direct measure of β0 and β2, the topological variables that quantitate both the number of isolated bone fragments and the number of imbedded pores. For the interconnected cluster, the connectivity, β1, was determined by the relationship β1 = β0 − X + β2, where X is the Euler-Poincaré characteristic calculated with the method described by Feldkamp et al.(4) β 1, the topological connectivity, is an enumeration of the number of closed loops in the analyzed volume. We also calculated the 3D BV/TV from the 3D image analyzed (3D BV/TVx).
For specimens with comparable BV/TV, it was suitable to normalize the number of connections by dividing by the total analyzed volume, expressing the result as connections per total cubic millimeter of trabecular bone. For this study, in which BV/TV was 50% lower in OVX rats than in sham-OVXd rats, we normalized the number of connections by dividing by the 3D BV/TV in the region, giving connectivity per cubic millimeter of trabecular bone (β1/3D BV/TVx).(7,8) The coefficient of variation (CV) for trabecular connectivity expressed as connectivity per cubic millimeter of trabecular bone ranged from 7–13% in the sham-operated animals and from 10–26% in the OVXd animals for the XTM technique.(8)
Biochemical tests and 17β-estradiol assays
Urine samples were stored at −20°C until analyzed in duplicate by enzyme-linked immunosorbent assay (ELISA) for deoxypyridinoline (Dpyr) cross-links (Metrabiosystems, Mountain View, CA, U. S.A.). The ELISA has a CV of 5–10% in our laboratory and has good accuracy compared with standard high-performance liquid chromotomgraphy.(27,28) Corp., Los Angeles, CA, U.S. A.). Intra-assay and interassay CVs ranged from 4–8%.
Histomorphometry of the left proximal tibia
The proximal centimeter of the left tibia was sawed off and prepared for histomorphometric analysis by standardized methods.(29,30) Pairs of 5-μm frontal sections were prepared from the anterior aspect with a Richert-Jung Supercut 2050 Microtome (Heidelberg, Germany). The first was stained by the Goldner method,(31) and the second was left unstained.
A counting window, measuring 7.2 ± 0.9 mm2 and containing only cancellous bone and bone marrow, was created for the histomorphometric analysis.(32) With the use of the camera lucida projecting onto a digitizer, areas, perimeters, and distances as below were digitized using BIOQUANT II software (R & M Biometrics, Nashville, TN, U.S.A.). The raw data were used to derive calculated histomorphometric endpoints.
With the light microscope, total tissue area (Tt.Ar), Tb.Ar, and bone surface (Tb.Pm) were measured. Osteoclast surface (Oc.Pm) was measured from the Goldner-stained slide at × 400. With epifluorescent illumination, single-labeled surface (sL.Pm) (×160), double-labeled surface (dL.Pm) (×160), and interlabel thickness of double labels (IrL.Wi) (×400) were measured from the unstained slide.
Histomorphometric variables representing the following bone features were calculated: BV/TV and structure, mineralizing surface (MS/BS), individual osteoblast activity, and Oc.Pm. From Tt.Ar and Tb.Ar, BV/TV, Tb.Th, Tb.Sp and Tb.N were calculated.(33) From Tb.Pm, dL.Pm, and Oc.Pm, both the percentage of double-labeled surface and the percentage of MS/BS (based on double plus half-single label), and percentage of osteoclast surface (OcS/BS) were calculated. From IrL.Wi and the interlabel time period, the mineral apposition rate (MAR), a measure of individual osteoblast activity, was calculated. Finally, surface-based, bone volume-based, and total tissue volume-based bone formation rates (BFR/BS, BFR/BV, and BFR/TV) were calculated.(33-35)
The mean and SDs are reported for each time point studied for animal weight, BV/TV, trabecular bone connectivity, and other histomorphometric variables, Dpyr cross-links and 17β-estradiol levels. To determine differences at the separate time points for 3D BV/TV, trabecular bone connectivity, histomorphometric variables, and Dpyr cross-link excretion, repeated measures analysis of variance was done. Tukey's post hoc tests were done to identify time points that were significantly different (p < 0.05).(36) Also, a repeated measures analysis of variance with orthogonal contrasts was done to determine if a significant trend was present for changes in trabecular bone volume and trabecular BV/TV thickness from the time of ERT initiation.(36)
All animals tolerated surgery and scanning without complications. Animals showed modest weight changes during the study (Table 1).
Table Table 1.. Study Group Descriptions, Weight Changes at Day 0 and Day 50, and 17β-Estradiol Levels at Day 50
Measurements of BV/TV and structure by XTM (BV/TVx and β1/BV/TVx)
Trabecular bone volume (BV/TVx) and connectivity per cubic millimeter of trabecular bone (β1/BV/TVx) measured by XTM were maintained in the sham-OVXd and OVX animals given ERT from the time of OVX. However, OVX + vehicle–treated animals lost 54% BV/TVx and 46% β 1/BV/TVx from baseline (p < 0.01 (Figs. 1 and 2). Though reduced BV/TVx and β1/BV/TVx values were seen in animals rescanned at 5–8 days post-OVX, this reduction was only significant by day 13 post-OVX, reaching 22% and 25% (p < 0.01) below baseline values, respectively. Initiating ERT at days 5, 8, and 13 post-OVX restored BV/TVx to baseline values by day 50. However, β1/BV/TVx, at day 50 was significantly less than baseline values in animals treated with ERT from days 5, 8, and 13 (p < 0.01). The change in BV/TVx from the initiation of ERT to day 50 was significantly greater in all ERT-treated animals compared with sham-OVXd animals (p < 0.05) (Fig. 1).
Tb.Th measured from the 3D images increased by 10–20% of baseline values in all ERT-treated groups, although the mean increase in Tb. Th was not significantly different among groups at day 50 (Table 2). However, in animals that had three serial XTM scans, the initiation of ERT (at days 5, 8, and 13) resulted in a significant increase in Tb.Th at day 50 when analyzed for a trend (p < 0.005). A time sequence for trabecular bone structure of a group 2 (OVX + vehicle) and group 6 (OVX + ERT from days 13–50) animal is shown in Fig. 3.
Table Table 2.. Changes in Tb.Th, Tb.N, and Tb.Sp. Measured by XTM (%) from Day 0 to Day 50 Post-OVX
Tb.N decreased 50% from baseline in OVX + vehicle–animals (Table 2) (p < 0.05) but did not change significantly in any other group. Trabecular spacing increased 5-fold from day 0 to day 50 in OVX animals treated with vehicle (p < 0.05).
Measurements of bone turnover
After 50 days post-OVX (OVX + vehicle), bone formation endpoints (MS/BS, MAR, and BFR/BS) (Table 3) were significantly higher (p < 0.05) than sham-OVXd animals. OcS/BS in OVX animals was double that of sham-OVXd animals (p < 0.05) (Table 4). At day 50, all ERT animals had bone turnover endpoints (MAR, OcS/BS, MS/BS, and BRF/BS) that were significantly lower than OVX + vehicle animals (p < 0.05). All ERT-treated animal's bone resorption marker (OcS/BS) was similar to sham-OVXd; however, bone formation markers (MAR, MS/BS, and BRF/BS) were significantly higher (p < 0.05) than sham-OVXd animals at day 50.
Table Table 3.. Effect of OVX on Trabecular Bone Structure, Bone Formation, and Resorption Endpoints by Standard Histomorphometry
Table Table 4.. Changes in Dpyr Cross-Link Excretion (nMDPD)/mMCr) from Day 0–50 Post-OVX
Bone resorption measured by serial determination of Dpyr cross-links (Table 4), increased 22% by day 5 post-OVX, 40% by day 8, 50% by day 13, and about 100% by day 50 over baseline values (p < 0.05 from day 0 post-OVX). ERT initiated at days 0, 5, 8, and 13 post-OVX was associated with reduced Dpyr cross-link excretion to baseline values.
17β-estradiol levels (Table 1) measured at day 50 in sham-OVXd animals were 16.5 ± 6.1 pg/ml and in OVXd animals treated with vehicle were undetectable (p < 0.05 from all other groups). Animals given ERT had 17β-estradiol levels similar to sham-OVXd animals.
Estrogen deficiency resulted in both a loss of trabecular bone and in the deterioration of trabecular connectivity. Serial, 3D in vivo measurements of trabecular structure by XTM revealed a decrease in trabecular bone connections and bone volume within 5–13 days of OVX that continued until day 50. ERT initiated at the time of OVX maintained trabecular bone connectivity and bone volume. We also observed that the decrease in BV/TV was restored by day 50 when ERT was initiated within 5–13 days of OVX. The restoration of trabecular bone mass was associated with modest thickening of trabeculae.
Loss of trabecular bone connectivity occurs rapidly with estrogen deficiency. Other investigators have observed an increase in the number of osteoclasts on the trabecular bone surface by 5–6 days post-OVX and significant reductions in trabecular bone mass within 9–10 days of estrogen deficiency.(9,13,20) Our results agree with those of the other studies, because BV/TV and connectivity had decreased significantly from baseline values by 13 days post-OVX. When ERT was initiated at days 5, 8, and 13 post-OVX, BV/TV but not connectivity was restored to baseline levels by day 50, indicating that changes in trabecular structure not reversible by ERT occurred by day 13 post-OVX.
We believe that the loss of BV/TV and connectivity observed with acute estrogen deficiency represents the increase in remodeling space that occurs when the bone turnover rate accelerates.(9,14,16,37) While the remodeling space in adult humans is simply observed as transiently decreased bone density,(11,17,21,37,38) it is observed in the rat as a transient decline in both trabecular connectivity and bone volume.(6-8,10,16) Since rat metaphyseal trabeculae are thinner than human trabeculae,(7-8,14,18,34,37) they are more likely to perforate during periods of increased activation frequency. This would result in a loss of connectivity and Tb.N. When ERT is initiated during the time when estrogen-depletion bone loss is due only to increased remodeling space (day 10–20 post-OVX), the decreased birthrate of new remodeling units not only decreases the perforation of the trabeculae but also allows existing trabeculae to fill in as ongoing remodeling units complete their formation phase.(9,39) This results in full restoration of bone mass.
In our experiment, ERT initiated at the time of OVX maintained trabecular connectivity and bone volume; however, when ERT was delayed from 5–13 days post-OVX, trabecular bone mass was restored to baseline values but connectivity was not. Shen et al. also administered ERT to OVX rats from the day 0–30 post-OVX and reported ERT maintained trabecular bone structure and trabecular connectivity.(16) However, animals that were OVXd and had ERT initiated from day 21–49 post-OVX had 20% lower BV/TV and trabecular connectivity than sham-OVXd animals, but BV/TV and connectivity was double that of OVX + vehicle–treated animals. Also, Abe et al. treated osteopenic OVXd rats with ERT alone or in combination with other antiresorptive agents and did not find an increase in BV/TV.(13) Therefore, our results suggest that experiments in the OVXd rat that are designed to document the ability of an agent to block bone loss and preserve trabecular structure should begin the day after OVX. “Preventive” treatments are occasionally delayed as long as 7–10 days.(13,16,39) Our results and those of others(8-9,13,16) suggest that the early occurrence of bone loss and apparent permanent loss of connectivity make such a delay in treatment something other than a true prevention study and could compromise the ability of detecting maximal agent effectiveness.
Estrogen and other antiresorptive agents, i.e., bisphosphonates and calcitonin, prevent bone loss mainly by reducing the rate of bone turnover, as assessed by histomorphometric measurements(9,13-16,18) and biochemistry.(40-43) OVX results in a significant increase in the activation frequency of bone remodeling units,(7-9,13,16,20,39) as evidenced by increased osteoclasts per bone surface, increased mineralizing surface, BFR/BS, and, to a lesser extent, MAR. Estrogen, bisphosphonates, and calcitonin reduce the activation frequency of new bone remodeling units as assessed by reduced OcS/BS, mineralizing surface, and BFR/BS compared with OVXd animals.(12-16,18,19) However, even when activation frequency is reduced, in the early phase of ERT bone formation rate remains above baseline levels.(13,18) These results suggest that the observed restoration of BV/TV resulted from continued bone formation, amidst a depressed bone turnover rate.(20) The accumulation of mild increases in bone mass observed with both types of agents suggest that mild positive bone balances persist for extended periods of time and have been associated with increased bone strength and a reduction in osteoporotic fractures.(19,44-48)
OVX was associated with a 100% increase in Dpyr cross-link excretion by day 50 post-OVX. ERT was associated with suppression of Dpyr cross-link excretion to baseline values in all treatment groups. In general, reduction of biochemical markers of bone turnover to premenopausal levels or below with estrogen or bisphosphonates is thought to be associated with maintenance of bone mass in postmenopausal women and in animal studies.(19,40-43)
There are some shortcomings in this study. First, the only site imaged for trabecular structure analysis was the proximal tibial metaphysis, an area that represents cancellous bone sites that lose bone rapidly after OVX. Other skeletal sites in the rat do not lose bone rapidly after OVX.(22,49) However, we used the proximal tibial metaphysis because not only does it lose bone post-OVX, but it is also the most common site reported in studies of estrogen-depletion osteopenia, so that we were able to compare our results to those of others. 17β-estradiol levels were within the normal range for intact rats; however, the serum was obtained only at the end of the study to measure 17 β-estradiol levels.(12,15) Therefore, the 17β-estradiol levels may not reflect the serum level during the study. Last, in this study, ERT suppressed bone formation rates and bone resorption endpoints significantly below that seen in the sham-OVXd rats. This suggests that our dose 10 μg/kg/day three times per week) may have caused rats to become over-replete with estrogen. Other studies have found this dose and dosing regimen of ERT in OVXd rats maintained trabecular bone mass in acute estrogen deficiency, but a dose response has never been reported.(14) Therefore, more dose response studies with ERT of this type are required to find an ERT dose that maintains bone volume but does not suppress bone turnover endpoints to below normal values.
In summary, we demonstrated that loss of trabecular bone and irreversible changes in trabecular connectivity occur rapidly with acute estrogen deficiency. When ERT was delayed 5–13 days post-OVX, declines in BV/TV and connectivity occurred. ERT restored BV/TV, but not connectivity, to baseline levels by allowing bone formation to continue in previously activated bone remodeling units while suppressing the production of new remodeling units. This may be the mechanism by which estrogen and other antiresorptive agents restore lost bone mass, and over time this leads to a reduction in the risk of osteoporotic fractures in postmenopausal women.
We would like to acknowledge Tom Breunig, Ph.D. and Daniel Witkin for their assistance in obtaining the XTM scans. The synchrotron work was performed at SSRL, which is funded in part by the U.S. DOE Basic Energy Sciences Division. LLNL is under contract W-7405-ENG48 with the U.S. DOE. This work was supported by Public Health Service Grants 1-R01-AR 43052 and the Rosalind Russell Arthritis Research Center.