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Keywords:

  • bone quality;
  • bone strength;
  • bisphosphonate treatment;
  • μCT;
  • non-human primates

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The dose-dependent effect of ibandronate treatment on bone mass and architecture was assessed in a large animal study of OVX monkeys using μCT for quantitative bone morphometry and biomechanical testing for measures of bone strength. The study showed that intermittent ibandronate preserved lumbar spine bone quality and strength in these animals after 16 months of treatment.

Introduction: Ibandronate is a bisphosphonate, which is a class of compounds that, in pharmacologically active doses, not only suppresses bone resorption and turnover but also prevents loss of bone mass and strength in the ovariectomized (OVX) rat.

Materials and Methods: We evaluated the effects of ibandronate on bone mass and architecture in the OVX cynomolgus macaque. Sixty-one adult female macaques were divided into five groups (N = 11–15): sham control, OVX control, and OVX low- (10 μg/kg), medium- (30 μg/kg), and high- (150 μg/kg) dose ibandronate. Treatment was administered by intravenous bolus injection every 30 days for 16 months starting at ovariectomy. This dosing schedule is equivalent to a 3-monthly dosing regimen in human subjects over 4 years. Animals were killed at the conclusion of the study, and excised bone specimens of the first lumbar vertebra (L1) were evaluated for quantitative bone densitometry, morphometry, and mechanical properties. Architectural parameters were assessed by μCT including direct 3D bone morphometry. A measure of specimen strength was obtained using destructive compression testing.

Results and Conclusions: A significant loss of bone mass and related changes in bone architecture after ovariectomy resulted in a reduction of whole bone strength as expressed by high correlations between architectural and mechanical properties. In this analysis, BMC was the best single predictor of whole bone strength (r2 = 67%). Nevertheless, including architectural indices in a multiple linear regression analysis increased that prediction to 88%. With respect to the treatment, the medium- and high-dose groups were not significantly different from the sham group for all bone mineral and structural parameters. Additionally, significant differences were seen for all measured parameters between the high-dose group and the OVX group, and for some parameters, between the medium-dose group and the OVX group. Intermittent ibandronate treatment effectively and dose-dependently prevented bone loss, architectural deterioration, and strength reduction in the lumbar spine of OVX monkeys.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bisphosphonates are a class of compounds that in pharmacologically active doses not only suppress bone resorption and turnover in estrogen-depleted rats, dogs, and monkeys, but also prevent loss of bone mass and bone strength.(1–13) Ibandronate, a highly potent nitrogen-containing bisphosphonate, is the only bisphosphonate that is under clinical development for both oral and intravenous administration with extended between-dose intervals in postmenopausal osteoporosis (PMO).(14–16) When administered orally in a daily regimen or intermittently with a between-dose interval of >2 months to women with PMO, ibandronate has been shown to reduce the risk of new vertebral fractures by 62% and 50%, respectively, significantly increase BMD, and reduce biochemical markers of bone turnover.(17) The robust antifracture results of the BONE study of oral ibandronate administered either daily or with a between-dose interval of >2 months proves the concept that intermittent ibandronate regimens, with between-dose intervals of >1 week, can significantly and substantially reduce fracture incidence.(17), (18) Consequently a large trial is ongoing to investigate a convenient and novel once-monthly regimen of oral ibandronate in postmenopausal osteoporosis.

Intravenous ibandronate injections given once every 3 months have also been shown to be effective in the management of PMO.(16), (19–21) Intravenous administration offers a useful alternative to oral administration for certain patients, for example, non-ambulatory patients who cannot adopt the upright posture mandatory for oral dosing, patients experiencing poor upper gastrointestinal tolerability with current oral bisphosphonates, or patients who may find the need to fast before and after oral dosing inconvenient. The availability of both oral and intravenous regimens for ibandronate will provide the clinician and patient with a choice, enabling treatment to be tailored to the individual's needs and circumstances.

Bisphosphonates have great affinity for the calcium phosphate on the surface of bone that is being formed or resorbed.(22) Once on the bone surface, bisphosphonates inhibit bone resorption, and because they inhibit bone formation to a lesser extent, they increase bone mass.(22) Studies of bisphosphonate efficacy focus on trabecular bone, where the turnover rate is much higher than in cortical bone.(22) The effects of bisphosphonates on Haversian (cortical) remodeling in the adult human skeleton are less well understood.(12), (23)

We recently showed that the overall skeletal effects of ibandronate on both cortical and trabecular bone seen in the non-human primate model have a high likelihood to closely mimic what can be expected in human clinical trials.(9) That study is thus far the only study in non-human primates that appropriately reflects the long-term intermittent intravenous dosing regimen intended for clinical use. Indeed, the World Health Organization(24), (25) has noted that many preclinical studies of the effects of antiresorptive or anabolic agents on bone conducted in appropriate animal models have accurately predicted their effects in humans. Of all models, the OVX non-human primate is therefore considered most appropriate in which to study effects on bone remodeling.(25–27)

This study is based on the hypothesis that the intermittent administration of ibandronate can prevent bone loss and the deterioration of bone architecture in the OVX macaque model. Therefore, the specific aims of the study were to first examine the effects of ovariectomy on the mass and structure of vertebral bone in the adult female cynomolgus macaque compared with sham controls and to provide baseline data for this estrogen-depleted osteoporosis model. Second, the various parameters of bone mass and architecture for the animals in the three different ibandronate dosing groups (10, 30, and 150 μg/kg IV at monthly intervals) were compared with those of the sham and untreated OVX groups to determine whether bisphosphonate treatment could prevent the effects of ovariectomy. While previously reported results of that study were related to the general beneficial effects of ibandronate on BMD, bone strength, biochemical markers of bone turnover, and 2D histomorphometry,(9) the specific aims of these analyses focused for the first time on the relation between bone strength, bone mass, and the 3D architecture in lumbar vertebrae of non-human primates, as measures of bone quality. Third, we wanted to investigate whether the assessed mechano-structure relationships were the same for normal, OVX, and OVX plus ibandronate-treated bone. The intravenous dosing schedule of ibandronate used in this study, once-monthly administration for 16 months, corresponds to a 4-year treatment with one injection every 3 months in human subjects.(25), (28)

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animal population

All animal material used in this study represents a subset of bones from a previously published study.(9) Briefly, for the study, 66 female cynomolgus macaques (Macaca fascicularis; >9 years of age) were divided into five groups. After an acclimation period and 2 months of baseline observation, during which all the animals were monitored, all groups underwent a surgical procedure. Group 1 had a sham ovariectomy, whereas groups 2–5 had both ovaries removed (OVX). The sham (group 1) and OVX (group 2) control groups (N = 15 each) received vehicle placebo, and the remaining three OVX groups (N = 12 each) received the vehicle and the study drug (ibandronic acid sodium monohydrate BM 21.0955 · Na · H2O [ibandronate]) at doses of 10, 30, or 150 μg/kg body weight (i.e., low- [group 3], medium- [group 4]. and high- [group 5] dose, respectively. Dosing started on the day of surgery, and all animals received intravenous bolus injections of vehicle or the vehicle and ibandronate every 30 days for 16 months. The three dose levels were chosen based on previous studies in estrogen-depleted rats and dogs and were expected to represent a suboptimal dose, the optimal effective dose, and five times the optimal dose, respectively. Animals were housed in groups of two or three, with free access to food and water and a 24-h light/dark cycle. Three animals died during the study; the deaths were considered unrelated to ibandronate treatment. At 16 months, 63 animals were killed; 2 OVX control animals were excluded from further analysis because of the presence of ovarian tissue at study termination. Use of non-human primates in this research project was reviewed and approved by local and governmental animal ethics committees.

Bone specimen preparation

The first lumbar vertebra (L1) of 61 animals was excised and immediately wrapped in saline-soaked gauze. Individual bone specimens were placed in labeled airtight plastic bags, and all specimens were stored at approximately −20°C.

After thawing, plain radiographs (Faxitron 43855A; HP, McMinnville, OR, USA) were taken of the L1 vertebrae in both the anterior/posterior (AP) direction and the superior/inferior (SI) direction to assess the specimens for gross defects before evaluation. The vertebrae were cleaned of excess soft tissue, and spinal processes were removed using a hand-held oscillating bone saw to isolate the vertebral body for imaging and mechanical testing.

Specimen evaluation

Whole specimen BMC (g) and BMD (g/cm2) were obtained using DXA (QDR 2000+; Hologic, Waltham, MA, USA) with specimens submerged in water and positioned on a 1-in-thick acrylic plate. Specimens were scanned in the AP direction using the small animal mode, and analysis was performed using the animal protocol subregion option. The vertebra AP diameter, measured with a hand-held pair of digital calipers, was used to normalize the DXA BMD values (width-adjusted DXA BMD = wBMD) for individual animal size differences. pQCT (XTC 960A; Stratec, Pforzheim, Germany) was used to measure the total sagittal cross-sectional area (TOT_A) at the vertebral body midpoint. This parameter was used to calculate the ultimate strength of the specimen in compressive loading, assuming failure at the midpoint of the vertebral body (Apparent Strength). Likewise a modulus was calculated with this area value for an assumed midpoint failure (Apparent Modulus).

The L1 vertebral bodies were measured using a micro-tomographic imaging system (μCT 20; Scanco Medical, Bassersdorf, Switzerland), a compact fan-beam type of tomograph, also referred to as desktop μCT. The system was designed for the noninvasive measurement and analysis of unprocessed surgical bone biopsy specimens and small animal bones.(29) For each sample, the entire extent of the vertebra was scanned using a voxel size of 34 μm3 in the cranial-caudal direction; this resulted in a total of 337–578 micro-tomographic slices, depending on the actual height of the individual vertebra (range, 11.5–19.7 mm). The specimens were totally submerged in saline during the measurement to prevent their drying out. Measurements were stored in 3D image arrays. A 3D Gaussian filter with a limited, finite filter support was used to partly suppress the noise in the volumes. In the next step, bone was segmented from background with the help of a global thresholding procedure.(30) Samples were binarized using the same parameters for the filter width (1.2), the filter support (1), and the threshold (196‰ of the maximum grayscale value). Subsequently, all samples were analyzed using both visual assessment and quantitative morphometry for two distinct regions: (1) the whole vertebra including both trabecular and cortical bone; (2) a trabecular volume of interest (VOI) that was defined as the maximal box that could be fitted within the trabecular part of the vertebral body. The visual assessment consisted of both 2D (orthogonal cut planes in transverse and coronal directions) and 3D representations.(31) Morphometric and architectural indices were determined from the micro-tomographic examinations of the intact specimen as well as the trabecular VOI. BMC based on the measured bone volume (BV), and the assumption of a constant tissue density was analyzed for the intact vertebrae, and bone volume density (BV/TV), bone surface density (BS/TV), and specific bone surface (BS/BV) for the trabecular VOI were determined directly from the respective binarized volumes. Trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were calculated using a direct, model-independent 3D approach.(32) In addition, the structural anisotropy in the spherical VOI was determined using mean intercept length (MIL) measurements.(33) MIL denotes the average distance between bone/marrow interfaces and is measured by tracing test lines in different directions in the examined VOI. From this measurement, a MIL tensor was calculated by fitting the MIL values to an ellipsoid.(33) The eigenvalues of this tensor ( H1 - H3 ) were used to define the degree of anisotropy (DA), which denotes the maximum to minimum MIL ratio.(34) Because we expected the analyzed specimens to show distinct differences in bone tissue connectivity, connectivity was also computed for the trabecular VOI based on a 3D extension of the trabecular bone pattern factor (TBPf).(35) Additionally, the structure model index (SMI), a new parameter for the direct characterization of the model type (plate-like versus rod-like) of a given bone architecture, was calculated.(36)

After imaging, the specimens were subjected to destructive compression testing to determine whole bone ultimate load, stiffness, and apparent strength and apparent modulus at the midsection. A servo-hydraulic material testing machine (Instron Model 8511; Instron, Canton, MA, USA) was used to displace an upper loading platen at a strain rate of 0.5%/s (based on the average vertebral body height) until specimen failure occurred. PMMA (poly-methylmethacrylate, FASTRAY Dental Cement; Bosworth Co., Skokie, IL, USA) end caps, made with impressions of the superior and inferior ends of the L1 vertebral body, created flat loading surfaces on the specimens. Acquisition software (Labview, v 3.0.1; National Instruments, Austin, TX, USA) was customized to generate a load-versus displacement-curve to determine ultimate load (N) and stiffness (slope of linear elastic region of the curve, N/mm). Apparent strength (N/mm2) and apparent modulus (N/mm2) were calculated using the sagittal cross-sectional area at the midpoint.

Statistical analysis

Group variance homogeneity was assessed using Levene's test (0.001 level of significance). Whenever group variances were found to be homogeneous, group means were compared within a one-factor ANOVA. If a significant group effect was found, the sham and OVX control groups were compared, and each dose group was compared against the sham control group as well as the OVX control group using one-factor ANOVA and Fisher's PLSD least significant difference posthoc analysis at the 0.05 significance level. Dose-response relationships were evaluated when a significant group effect was found. The OVX control group and the treated groups were used to assess the significance of a linear and/or quadratic trend across dose levels (0.05 level of significance). BMDP software (Statistical Solutions, Saugus, MA, USA) was used to analyze groups for dose-dependent trends as well as for single- and multiple-regression analyses to predict ultimate load from bone mass and/or bone architecture. Multiple linear regressions (MRLs) were used to model ultimate load using structural parameters as independent variables.(37) The regression analysis produced a coefficient of multiple determination r2, which represents the proportion of variability in the dependent parameter explained by the structural parameters in the model. In the MRL analysis, BMC was always included as the first parameter, and additional structural parameters were included starting with the parameter with the highest significant (0.05 level) contribution to a better determination of ultimate load and ending when the increase in the coefficient of determination was no longer significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The results reported in the text are those identified by the statistical analysis as significantly different from either sham or OVX controls; all other parameters did not differ significantly from those of the sham or OVX groups. The complete DXA, μCT, and mechanical test results are presented in Table 1.

Table Table 1.. 3D Mass and Morphometric Indices from μCT as Well as Biomechanical Properties of Lumbar Vertebrae in Compression
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DXA scanning

Ovariectomy resulted in significantly lower L1 vertebrae DXA BMD (−16%) and wBMD (−18%) compared with the sham group. The BMD and wBMD of the low-dose ibandronate group were also significantly lower (−12% and −13%) than those of the sham group. Compared with the OVX group, DXA wBMD was significantly higher (+12%) in the medium-dose ibandronate group, whereas DXA BMC (30%), BMD (23%), and wBMD (23%) were all significantly higher in the high-dose group. Significant linear trends for increasing DXA, BMC, BMD, and wBMD were identified across the dose groups (Table 1).

μCT evaluation

Ovariectomy resulted in a significantly lower L1 vertebra BMC (−14%) than in the sham group. BMC was also significantly lower in the low-dose ibandronate treatment group (−18%) compared with the sham group, but significantly higher (+23%) in the high-dose ibandronate group than in the OVX group. There was a significant linear trend for increasing μCT BMC across the dose groups. These results are presented graphically in Fig. 1A (Table 1).

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Figure FIG. 1.. (A) Groups 2 (OVX) and 3 (low) had significantly lower BMC compared with group 1 (sham). Group 5 (high) was significantly higher compared with the OVX group. (B) Ultimate load is significantly decreased in the OVX and low-dose group compared with sham, and the high-dose group is significantly increased compared with the OVX group. (C) Changes in Tb.Sp and Tb.Th as measured with OVX and treatment.aSignificantly different from sham controls at p < 0.05;bsignificantly different from OVX controls at p < 0.05.

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In the trabecular VOI, ovariectomy significantly decreased or increased all determined structural indices (range, −13% to 83%) except for BS/BV, which seemed unaffected by either ovariectomy or treatment. In the low-dose ibandronate group, structural properties were similar to those of the OVX group, although BV/TV (−13%) and SMI (+83%) showed significant differences compared with the sham group. In the medium-dose ibandronate group, bone loss and accompanying deteriorations in bone architecture were almost entirely prevented apart from a relatively small decrease in Tb.Th (−11%), which was significantly different from that in the sham group. BS/TV, Tb.N, and Tb.Sp differed significantly from those of the OVX group and achieved values similar to those of the sham group. Although BV/TV, SMI, TBPf, and DA in the medium-dose group did not differ significantly from the OVX group, neither did they differ significantly from the sham group. In the high-dose group, all structural indices were similar to those of the sham group (range: −1% to 14%) and differed significantly from those of the OVX group. Significant linear trends were identified for all the structural indices except BS/BV, Tb.Th, Tb.Sp, and SMI. The between-group differences in Tb.Sp and Tb.Th are presented graphically in Fig. 1C.

2D and 3D visualizations of trabecular bone architecture were obtained to visualize architectural changes associated with ovariectomy and treatment (Fig. 2). Representative animals, defined as those with median bone volume density, were selected from groups 1 (sham), 2 (OVX), 4 (medium ibandronate dose), and 5 (high ibandronate dose). Results from the median animals in group 3 (low dose) are not shown because of the small differences found qualitatively and quantitatively in that group compared with group 2 (OVX). The transition from the clearly plate-like structures as seen in the sham group to a more rod-like structure in the OVX group is evident. The figure also shows that ibandronate treatment clearly prevented these changes.

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Figure FIG. 2.. 2D and 3D trabecular bone architecture of four median lumbar vertebrae (L1). Measurements made noninvasively using a micro-tomographic fan-beam imaging system with a resolution of 34 μm.

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Figure 3 shows the heterogeneity in bone architecture as shown in a series of transverse sections of a single vertebra (2–12 mm below the superior end plate). In this representative OVX control animal, the bone architecture close to either end plate is more isotropic and denser, whereas the structure in the middle of the bone is more clearly oriented in the AP/SI direction and is more porous. This trend can also be observed across different groups as depicted in Fig. 2 for coronal sections of the vertebrae. The middle zone is always less dense and more oriented, whereas the structure toward the end plates becomes denser and more isotropic. In the OVX group, it seems that the structure becomes more anisotropic with missing struts in the transverse direction. This could also be shown quantitatively with the degree of anisotropy being significantly increased in the OVX group. This was reversed by ibandronate treatment in a dose-dependent fashion.

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Figure FIG. 3.. Heterogeneity in bone architecture in a single lumber vertebra (L1) of a representative OVX animal in the cranial-caudal direction. The vertebra was measured noninvasively using 3D micro-tomographic imaging providing a nominal isotropic resolution of 34 μm.

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Additionally, the two modalities (DXA and μCT) used to assess BMC were found to be highly correlated (R2 = 0.96; p < 0.0001; Fig. 4), and the average values from both techniques did not significantly differ from each other.

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Figure FIG. 4.. Correlation between DXA- and μCT-derived BMC. Where DXA is able to measure true absorption coefficients and convert them to a bone content measured in grams, μCT-derived BMC is calculated from bone volume with the assumption of a constant bone tissue density. The high correlation and the slope of 1 show that this assumption is valid for this model of postmenopausal osteoporosis and within the limits of the measurement precision of both techniques.

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Mechanical testing

Ovariectomy significantly lowered both compressive ultimate load and corresponding apparent strength by 25% compared with the sham group. Ultimate load and apparent strength were also significantly lower in the low-dose group than in the sham group (−24% and −23%, respectively) but significantly higher (+32% and +31%, respectively) in the high-dose group than the OVX group. There were significant linear increasing trends across the dose groups for both ultimate load and apparent strength. The between-group differences in ultimate load are presented graphically in Fig. 1B (Table 1).

We were also interested how changes in bone mass and architecture as a measure of bone quality affected the mechanical competence of bone. Single regression analysis of the mechano-structure relationship between ultimate load and densitometric and architectural parameters revealed that, as a single parameter, μCT-derived BMC was most important in the prediction of ultimate load (r2 = 67%) followed by SMI (r2 = 56%) and BS/BV (r2 = 55%). In the multiple linear regression analysis, Tb.Sp, SMI, and BS/BV contributed an additional 21% independently of BMC (p < 0.05), explaining a total of 88% of the mechano-structure relationship (Table 2; Fig. 5). Even more interesting was the relative importance of bone mass and architecture when we looked at the individual groups (Fig. 6). Where in the sham group BMC was the single best predictor (BMC r2 = 84%; Tb.Sp r2 = 5%; SMI r2 = 40%), this was changed in the OVX group, where Tb.Sp performed better (BMC r2 = 19%; Tb.Sp r2 = 78%; SMI r2 = 12%). In the treated group, the importance of BMC in predicting ultimate load increased with dose (low r2 = 17%; middle r2 = 74%; high r2 = 87%). Nevertheless, where in the OVX group Tb.Sp outperformed all other parameters, in the treated groups, Tb.Sp made no significant contribution (r2 = 1–8%), and the contribution of SMI increased with increasing dose (r2 = 38–83%), clearly suggesting that there are different mechano-structure relationships for normal, diseased, and treated bone. Although bone mass was often the best single predictor, bone architecture played a significant role in the assessment of mechanical bone status, especially in the low-density regimen (OVX).

Table Table 2.. Coefficients of Determination for Single- and Multiple-Regression Analyses for the Prediction of Ultimate Load From μCT-Derived Bone Indices
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Figure FIG. 5.. (A) Fitted vs. measured ultimate load using BMC as a single predictor and (B) the ultimate load fitted from BMC plus three independent structural indices including Tb.Sp, SMI, and specific BS/BV. Inclusion of architectural properties resulted in a reduced spread of the data around the regression line, leading to a more accurate prediction of the measured ultimate load.

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Figure FIG. 6.. Effect of OVX and treatment in group-wise linear regression analyses for ultimate load with BMC, SMI, and Tb.Sp, respectively, as independent variables.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Osteoporosis, which occurs most frequently in postmenopausal women and elderly individuals of both sexes, is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture. Bone strength reflects the integration of two main features: BMD expressed as grams of mineral per area or volume and bone quality referring to bone architecture, turnover, damage accumulation, and mineralization.(38) It was hypothesized that the osteoclast-inhibiting bisphosphonate, ibandronate, would prevent bone loss and the deterioration in structural properties that result from ovariectomy in the cynomolgus macaque.

This study evaluated the effects of ibandronate on bone mass, architecture, and mechanical properties in the OVX macaque. The 3D micro-tomographic imaging device used in this study significantly extended the noninvasive evaluation of the bone specimens. Whereas the DXA measurements provided only 2D whole bone mineral information, μCT collected 3D information on whole and trabecular bone parameters in a single measurement.

After ovariectomy, densitometry and mechanical parameters in the OVX control group were significantly decreased compared with the sham control group. Morphometrically assessed parameters showed a deterioration of the underlying bone in the OVX control group. Ibandronate treatment dose-dependently prevented the effects of ovariectomy. In the low-dose group, only 3 (BMC, BV/TV, SMI) of the 10 parameters evaluated differed significantly from the sham group. In the medium-dose group, 3 (BS/TV, Tb.N, Tb.Sp) of 10 were significantly different from the OVX group, and only 1 (Tb.Th, see below) was significantly different from the sham group. In the high-dose group, all 10 parameters were significantly different from the OVX group. Nevertheless, the medium dose (30 μg/kg) can be considered to be an optimal effective dose to prevent OVX-induced changes in bone structure because results were not significantly different from sham controls. In this respect, results for the medium dose corresponding to a daily dose of 1 μg/kg were comparable with those for the defined optimal dose in estrogen-depleted rats(5), (6) and dogs.(8)

While previously reported results from this study described the general beneficial effects of ibandronate on BMD, bone strength, biochemical markers of bone turnover, and 2D histomorphometry,(9) the specific aims of these analyses focused for the first time on the relation between bone strength, bone mass, and the 3D architecture in lumbar vertebrae of non-human primates, as measures of bone quality. Nevertheless, the architectural data from this study were in good agreement with the previous study. There, 2D histomorphometric indices were assessed in the fourth lumbar spine from the same animals. Whereas 2D measures such as BV/TV and Tb.Th followed the exact same trends as their 3D counterparts in this study, these findings were not significant for 2D histomorphometry, although they were significant for 3D μCT. This is most likely because of the relatively small volume assessed in 2D histomorphometry compared with 3D μCT, reducing the precision and increasing the variability of the analysis. Nevertheless, the advantage of histomorphometry is not so much the assessment of static structural indices as the measurement of dynamic indices such as activation frequency, bone formation rate, or osteoclast surface to bone surface ratio. Those results showed clearly and significantly that OVX increased activation frequency and ibandronate treatment reduced activation frequency even beyond the sham controls in this model. For a more complete presentation of various indices in different bones, also including biochemical markers, we would like to refer to the original study.(9)

BMC measurements, whether by DXA or μCT, examine the whole bone specimen, which includes both trabecular and cortical bone material. However, it is known that the turnover rate in trabecular bone is much greater than in cortical bone, and the more rapid or more dramatic changes that may be occurring in the trabecular bone can be masked by the higher-density, less metabolically active cortical bone material. Therefore, it is vital to be able to examine trabecular bone in the specimens separate from the cortical bone. μCT imaging allows noninvasive examination of the internal structure of bone to determine the quantity, orientation, and connection of trabecular bone elements by focusing on a selected VOI containing only trabecular bone.

BMC as assessed from μCT was very similar to that of whole bone mineral measurements by DXA (Table 1; Fig. 4). This reaffirms that the amount of bone tissue is strongly related to its load-bearing capacity (Fig. 5A). Nevertheless, the mechanical strength of a bone specimen is also influenced by its internal structure. Of the architectural parameters, both Tb.Th and Tb.Sp correlated with ultimate load; however, only the changes in Tb.Sp were sufficiently large to account for the changes in ultimate load. Therefore, the decrease in ultimate load that accompanies ovariectomy seems primarily to result from the removal of individual elements, as well as the fenestration of existing plates. The shift in the SMI from a relatively low value characteristic of plates to those more indicative of rods supports this interpretation. In our MRL model, it was exactly those parameters (Tb.Sp, SMI), as well as the specific bone surface, that contributed to a significant increase in the prediction of ultimate load; from 67% for BMC alone to 88% including additional structural parameters (Fig. 5B).

In addition to these quantitative indices, the changes in bone architecture were also nicely shown using 2D and 3D visualizations of the apparent bone structure in the L1 vertebrae to assess bone status more qualitatively. The loss in bone mass is a clear consequence of thinning of individual trabeculae, leading to focal perforations in the structure and a resulting increase in trabecular spacing. High-dose administration of ibandronate fully prevented these effects. The medium dose of ibandronate clearly prevented most of the perforations, because trabecular spacing differed significantly from the OVX group but not from the sham group. Nevertheless, some concomitant thinning of the trabeculae remained evident in the medium-dose group.

Our findings from the regression analysis reveal that, in pooled populations, bone mass (BMC) is the single most important predictor for ultimate load and that, by including structural parameters such as trabecular spacing and SMI, the prediction can be improved significantly (Fig. 5). Nevertheless, when evaluating the mechano-structure relationships separately in normal, diseased, or treated groups, we find varying associations between ultimate load and the structural properties expressing the underlying biological changes in the bone architecture. From this analysis, it is interesting to see that, in the sham, mid, and high group, the contribution of BMC to bone strength is even higher (∼80%) than in the pooled group, whereas for the OVX group, BMC was not a good predictor (r2 = 19%), and Tb.Sp picked up 78% of the variance in bone strength. Knowledge about group-specific mechano-structure relationships might allow better prediction of individual fracture risk as well as the design of pharmacologic interventions best suited to restore normal bone properties.

The decrease in trabecular bone mass and the associated changes in the architectural properties caused by ovariectomy were as expected in this study, because the loss of estrogen after ovariectomy increases bone resorption more than bone formation.(39) The effect is more marked in trabecular bone, where the rate of turnover is much greater than in cortical bone.(22) However, there were clear trends toward reduced bone loss and less architectural deterioration with increasing doses of ibandronate. The mean values of most of the parameters of bone mass, architecture, and strength were similar to those of the sham group in the animals receiving the medium dose of ibandronate, whereas those animals in the high-dose ibandronate group were significantly higher than the OVX group and similar to, or sometimes above, those of the sham group. The dose dependence can be attributed to a greater inhibition of remodeling activity with higher doses. Similar trends were found in a recent mini-pig study using μCT to evaluate the changes in bone mass and structure with ovariectomy and risedronate treatment.(37) In that study, the authors also found that inclusion of multiple parameters in the prediction of ultimate load significantly increased the coefficient of determination from 76% to 91%. Nevertheless, where their study reported only on results for trabecular bone specimens, this study was able to show for the first time that the inclusion of bone architectural indices was also beneficial for the prediction of whole bone mechanical competence.

In conclusion, the results of this study show a significant loss of bone mass and related deterioration of bone quality as assessed from architectural properties after ovariectomy in the adult female macaque. Intermittent ibandronate effectively, and dose-dependently, prevented bone loss and architectural deterioration in the OVX animals. For all structural parameters except one (Tb.Th), the medium- and high-dose groups did not differ significantly from the sham group. Additionally, significant differences were seen for all measured parameters between the high-dose group and the sham group and for some parameters between the medium-dose group and the OVX group.

In this preclinical study, normal bone strength and bone quality were maintained with intermittent ibandronate. These results therefore provide further evidence to support the efficacy and safety of intermittent administration of ibandronate in the management of PMO. Furthermore, these findings are relevant to the ongoing optimization of intermittent ibandronate regimens in the clinical setting.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was partly funded by the SNF Professorship in Bioengineering of the Swiss National Science Foundation (FP 620–58097.99). The authors thank Jon Conta for performing biomechanical testing, Kristy Salisbury for assistance with the μCT measurements, and Dr Ralph Schimmer for helpful discussions during the preparation of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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