Detecting early bone changes using in vivo micro-ct in ovariectomized, zoledronic acid-treated, and sham-operated rats

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1 Osteoporos Int (2010) 21: DOI /s z ORIGINAL ARTICLE Detecting early bone changes using in vivo micro-ct in ovariectomized, zoledronic acid-treated, and sham-operated rats E. Perilli & V. Le & B. Ma & P. Salmon & K. Reynolds & N. L. Fazzalari Received: 17 June 2009 / Accepted: 9 September 2009 / Published online: 7 October 2009 # International Osteoporosis Foundation and National Osteoporosis Foundation 2009 Abstract Summary This study monitored in vivo the effect on bone microarchitecture of initiating antiresorptive treatment with zoledronic acid in rats at 2 weeks following ovariectomy, an early phase at which major degenerative bone changes have been found to occur. The treatment still facilitated the full reversal of cancellous bone loss in rat tibia, highlighting the importance of the time point of initiation of antiresorptive treatment. Introduction Injection of zoledronic acid in rats at time of ovariectomy has been found to fully preserve tibial bone microarchitecture over time, whereas injection at 8 weeks after ovariectomy has shown partial bone recovery. This study investigated the effect on microarchitecture of initiating antiresorptive treatment in the early phase following E. Perilli (*) : V. Le : B. Ma : N. L. Fazzalari Bone and Joint Research Laboratory, Surgical Pathology, SA Pathology and Hanson Institute, Frome Road, Adelaide 5000, Australia egon.perilli@imvs.sa.gov.au E. Perilli : V. Le : B. Ma : N. L. Fazzalari Discipline of Pathology, University of Adelaide, Adelaide, Australia E. Perilli Vision Lab, Department of Physics, University of Antwerp, Antwerp, Belgium P. Salmon Skyscan NV, Kontich, Belgium K. Reynolds School of Computer Science, Engineering & Mathematics, Flinders University, Adelaide, Australia ovariectomy, at 2 weeks, a time point at which major degenerative changes in the bone have been found to occur. Methods Female Sprague Dawley rats were divided into ovariectomized group, ovariectomized group treated with zoledronic acid, and sham-operated group. In vivo micro- CT scanning of rat tibiae and morphometric analysis were performed at 0, 2, 4, 8, and 12 weeks after ovariectomy, with zoledronic acid treatment beginning 2 weeks after ovariectomy. Data were first analyzed with repeated measures analysis of variance (longitudinal study design) and then without repeated measures (cross-sectional study design). Results The ovariectomized group demonstrated dramatic bone loss, first detected at week 2. Conversely, at week 4, the zoledronic acid-treated group returned microstructural parameters to baseline values. Remarkable increases in bone parameters were found after 6 weeks of treatment and maintained similar to sham group until the end. The longitudinal study design provided earlier detection of bone changes compared to the cross-sectional study design. Conclusions Treatment with zoledronic acid as late as 2 weeks after ovariectomy still facilitates the full reversal of cancellous bone loss in the rat tibia. Keywords Bone structure. In vivo microcomputed tomography. Osteoporosis. Rat ovariectomy. Zoledronic acid Introduction Osteoporosis is a major health problem in the increasing elderly population. This disease is characterized by low bone mineral density and the deterioration of bone tissue, with a consequent increase in bone fragility and suscepti-

2 1372 Osteoporos Int (2010) 21: bility to fracture [1, 2]. Common clinical imaging methods assess this disease on the basis of bone mineral density measurements; however, they fail to detect the deterioration of cancellous bone microarchitecture. To detect these changes, usually a bone biopsy is harvested from the patient and histologically examined, which, however, gives only a two-dimensional representation of a three-dimensional (3D) structure. Recently, nondestructive 3D methods, such as microcomputed tomography (micro-ct) [3, 4] and high resolution peripheral quantitative computed tomography have become available [5, 6], giving further insight into these microarchitectural modifications. In osteoporosis studies, the rat model is often used to study bone loss over time due to ovariectomy, and the efficacy of drugs aimed to prevent this bone loss [7 12]. Until recently, most published research has utilized a cross-sectional study design. In these studies, groups of animals are euthanized at various time points, after being ovariectomized or given a drug treatment. The cross-sectional design was necessary, as the quantification of the microarchitecture was done in vitro, by either histology or in vitro micro-ct [7, 13 15], whereas in vivo micro-ct, which allows longitudinal study designs, was accessible only at synchrotron facilities [16 18]. More recent developments in micro-ct technology have made compact in vivo micro-ct systems available, making it possible to then conduct longitudinal studies on animals in laboratories [11, 19 22]. A big advantage of performing longitudinal rather than cross-sectional studies is that a comparatively small number of animals is needed for making comparisons, as each animal acts as its own control. This also has advantages from a statistical point of view, as it reduces the possibility of natural variability within a group concealing differences due to treatment [11]. In a recent in vivo study on ovariectomized and sham-operated rats, Boyd et al. re-examined the micro-ct data obtained in the longitudinal study design using a cross-sectional approach, showing that the longitudinal approach provided earlier detection of bone changes [11]. Zoledronic acid is a novel bisphosphonate used in antiresorptive therapies; histology and micro-ct testing has shown it to be highly effective in preventing bone loss in ovariectomized rats [9, 21, 23 25]. The capability of in vivo micro-ct to track the efficacy of antiresorptive therapy in reversing bone mass and microarchitecture deterioration in ovariectomized rats has been demonstrated [12, 16, 26]. Boyd et al. detected major bone degenerative changes in the proximal tibia within the first 3 months postovariectomy, and suggested that antiresorptive treatment of ovariectomized rats in this early time window may be able to preserve the existing architecture [11]. It has been found that injection with zoledronic acid before and at ovariectomy preserves the microarchitecture over time [9, 24]. Recently, Brouwers et al. compared these effects in the proximal tibia with those of zoledronic acid given at 8 weeks after ovariectomy [12]. In that work, the later treatment showed significant bone recovery (e.g., the percentage difference in tibial bone volume fraction at week 8 and week 16 compared to baseline, decreased from ca. 45% to 35%, respectively), but with lower effects compared to the treatment given at ovariectomy. However, the time point of the late treatment injection in Brouwers et al.'s study was 8 weeks after ovariectomy, the second half of the 3 months time window for intervention suggested by Boyd [11]. It remains to be investigated the effect on tibial bone architecture of starting this antiresorptive treatment in the first part of the 3 months time window, for instance after 2 weeks of surgery, an early time point at which major degenerative bone changes have already been found to occur [12, 15, 22]. This early time point could be important for recovery of bone mass. In humans, this could be seen analogous to a time window of early intervention in a first period after onset of menopause. The present study uses a compact in vivo micro-ct system to monitor the microarchitectural changes in the cancellous bone of the proximal tibia of three groups of rats in vivo: ovariectomized rats (OVX), ovariectomized rats having zoledronic acid treatment initiated at 2 weeks after surgery (OVX + ZOL), and sham-operated rats (SHAM). The primary aim is to measure in vivo the effects of induced bone loss and antiresorptive treatment with zoledronic acid started early (2 weeks) after ovariectomy over time, as well as the effects of normal aging in the sham-operated rats. Measurements will be taken within the first 3 months postovariectomy, that is, at time point 0 (baseline) and at weeks 2, 4, 8, and 12 after the surgery. The secondary aim is to compare the ability to detect structural bone changes using a longitudinal design with those using a cross-sectional analysis approach in a drug treatment study. This will be done in a way similar to the study of Boyd et al. where sham-operated rats and ovariectomized rats were analyzed, however here, with the addition of the drug-treated group. Thus, the present study makes this comparison in a drug treatment study, by having the zoledronic acid treatment group in the analysis. Methods Animals Twenty female Sprague Dawley rats were purchased from Adelaide University (Waite Campus) and housed three to four per cage (dimension cm). At the age of 2 months, the animals (average weight 185±8.7 g) were anesthetized with a peritoneal injection of ketamine/ xylaxine (1 ml/100 g) and underwent in vivo micro-ct

3 Osteoporos Int (2010) 21: scanning (week 0, baseline scan). They were then randomly divided into three groups; a sham surgery was performed on the first group (SHAM, N=7), while the second (OVX, N=7), and third (OVX + ZOL, N=6) groups underwent bilateral ovariectomy. During all experiments, the animals were provided ad libitum access to tap water and food pellets consisting of 19% protein, 0.67% phosphorous, 0.78% calcium, and 2,000 IU/kg vitamin D. All the animals were then scanned at week 2, 4, 8, and 12 after surgery. At week 2, zoledronic acid (Novartis, Basel, Switzerland) was injected into the peritoneal cavity (1.6 µg/kg) of the animals of the OVX + ZOL group, and continued on a weekly basis until week 11. The animals were treated according to the Australian code of practice for the care and use of animals for scientific purposes endorsed by the National Health and Research Council of Australia. In vivo micro-ct scanning Scanning was conducted using a cone-beam type in vivo micro-ct scanner (Skyscan model 1076, Skyscan, Kontich, Belgium). All the scanning was done with the animals under anesthesia (injection of ketamine/xylaxine, 1 ml/100 g). The right hind limb of the rat was placed into a cylindrical plastic holder, to position the limb at the scanning midline and to prevent movement of the limb during scanning. Only the scanned leg was exposed to X-rays, minimizing the effects of radiation on the animal. The acquisition settings were the following: X-ray source voltage 74 kvp, current 100 μa; a 1-mm thick aluminum filter was used for beam hardening artifact reduction. The pixel size was 8.7 μm for a 4,000 2,096 CCD detector array (total field of view of mm, side height). The exposure time was 4.7 s, the rotation step 0.8, with a complete rotation over 197 (i.e., 180 plus the angle due to the cone of the X-ray beam). The total scanning time was 20 min. During acquisition, the scanning region was centered on the proximal tibia, including the knee joint and extending distally along the tibial diaphysis. The cross-section images were reconstructed using a filtered back-projection algorithm (software NRecon, V 1.4.4, Skyscan, Kontich, Belgium). For each scan, a stack of 1,200 cross sections was reconstructed corresponding to a total reconstructed height of 10 mm (Fig. 1), starting from the knee joint and extending distally along the tibial diaphysis, with an interslice distance of 1 pixel (8.7 μm). The reconstructed images were of 2,000 2,000 pixels each, 8.7 μm pixel size, and were stored as 8-bit images (256 gray levels). Morphometric analysis of cancellous bone From the stack of cross-section images, a volume of interest (VOI) containing only cancellous bone was extracted for Fig. 1 Longitudinal cross-section image (10 mm in height) of the proximal rat tibia obtained by in vivo micro-ct (8.7-μm pixel size). The cancellous bone region containing the secondary spongiosa is shown (solid line, volume of interest 3 mm in height), over which the structural parameters were calculated morphometric analysis (software CT Analyzer V , Skyscan, Kontich, Belgium). The VOI started at a distance of 1 mm from the lower end of the growth plate and extended distally for 350 cross sections (3 mm in height, Fig. 1). To allow the calculation of the structural parameters, the images were segmented (thresholded) into bone and nonbone using a uniform threshold algorithm. The threshold value was found using calibrated thickness measurements. This was done by scanning a micro-ct phantom composed of four aluminum foils of 20, 50, 100, 250 μm (inserts), embedded in polymethymethacrylate (cylinder of 13 mm), and calculating the direct thickness of these aluminum inserts [27]. The direct thickness, calculated by using the local sphere-fitting method, gives the thickness of the structure calculated in 3D without structure model assumption [28]. The threshold value, that minimized the root mean square error in thickness from the nominal values of the inserts, was used as the threshold for the segmentation of the rat cancellous bone. Radiologically, this phantom resembles a bone structure surrounded by soft tissue, as the insert material and thicknesses are within the same density and thickness range as cancellous bone structures, while the polymethymethacrylate embedding resembles the soft tissue surrounding the bone [27]. For morphometeric analysis, the following structural parameters were calculated over each VOI of cancellous bone (using software CT Analyzer): bone volume fraction (BV/TV), direct trabecular thickness (Tb.Th*), direct trabecular separation (Tb.Sp*), trabecular number (Tb.N), and structure model index (SMI). The BV/TV was calculated using the marching cubes method [26, 29], calculating the volume of the bone itself (bone volume)

4 1374 Osteoporos Int (2010) 21: over the volume of the VOI (tissue volume; BV/TV=bone volume/tissue volume) [30]. The Tb.Th* and Tb.Sp* are 3D measures of the average thickness of the cancellous bone structure and the average diameter of the marrow cavities, respectively. Both were computed by using the local sphere-fitting method [28]. The Tb.N, the number of trabecular plates per unit length [30, 31], was calculated using the formula Tb.N=(BV/TV)/Tb.Th*. The SMI, a topological index, is calculated using a differential analysis of the triangulated surface of the structure, which gives an estimate of the ratio of the number of plates to the number of rods composing the 3D structure [27, 32]. The resulting values for the SMI range from 0 to 3; with 0 indicating an ideal plate like structure and 3 indicating an ideal rod like structure, and the values between signifying a mixed structure composed of both plates and rods. The reproducibility of the morphometric measurements was assessed. The right tibia of a dead rat was scanned five times, with the same scanner settings as during the in vivo experiments, taking the animal out and repositioning it into the scanner each time. The morphometric parameters were calculated as described previously, and the coefficient of variation (CV) determined. The reproducibility was high, with CV found to be less than 3% in Tb.Sp*, SMI, and Tb. Th*, less than 4% in Tb.N, and 4% in BV/TV. Statistical analysis The structural parameters had a normal distribution (Shapiro Wilk test, p>0.05 for all the examined parameters). For all the comparisons, differences were deemed to be statistically significant at p<0.05. For both the longitudinal and cross-sectional study design, analyses of the structural parameters were conducted to determine if (a) there was a time effect and (b) if there was a time by group interaction effect. The time effect indicates if there was a change over time within each group, and the time by group interaction effect indicates if different groups displayed different patterns of changes over time. Both study designs sought to (1) compare each variable measured at a certain time point with the corresponding baseline, (2) to compare each variable measured at a given time point with the previous time point, and (3) in the case of the OVX + ZOL group, also to compare the variables during treatment (week 4, 8, 12) with those at the time point of treatment start (week 2). The longitudinal study design employed a repeated measurement two-way analysis of variance (ANOVA) analysis over the structural parameters. If F values for a given variable were found to be significant, a paired Student's t test was used to investigate time-related changes. Conversely, the cross-sectional study design used a twoway ANOVA analysis. When F values were found to be significant, a Scheffe's post hoc test was used. Results From the initial 20 rats, 17 rats completed the study, whereas two OVX rats (#16, #18, respectively) and one OVX + ZOL rat (#20) died after week 8. Rats #16 and #18 died as the result of an adverse reaction to anesthetic, while rat #20 died from necrotized toes on the right foot. The in vivo micro-ct measurements taken at each time point allowed the monitoring of the structural changes in each rat (Table 1; Fig. 2). Figure 3 is a 3D representation of the micro-ct scans of a rat from each of the three groups, done at the different time points. In both the longitudinal and the cross-sectional study designs, both the time effect and the time by group interaction effect were significant for all structural parameters. Thus, each group showed significant changes with time, the patterns of which differed depending on the group. The SHAM group displayed a significant increase in BV/TV, Tb.N, and Tb.Th* (+145%, +70%, and +44%, respectively, at week 12 in relation to baseline; p<0.01; Table 1) as well as a decrease in SMI ( 13% at week 12 in relation to baseline; p<0.05). This indicates increased bone mass, accompanied by a transition from a merely rod-like structure towards a more mixed structure containing both plates and rods. In the longitudinal study design, the microarchitectural changes were detected as early as the second week; whereas in the cross-sectional study design, most of these changes were detected at a significantly later time point (BV/TV at week 4, Tb.N and SMI at week 12). Conversely, the OVX group showed a dramatic bone loss over time, with diminishing BV/TV, Tb.N and increasing Tb.Sp* ( 74%, 79%, and +284%, respectively, at week 12 in relation to baseline; p<0.05; Table 1; Fig 2). The SMI was found to increase (+14% and +15%, for week 4 and 8 compared to baseline, p<0.05), with week 12 being 10% higher than the baseline, but not reaching statistical significance (p=0.08). It is probable that this last result is due to a statistical artifact, produced by the reduced number of animals at the last time point (from seven to five, with the deaths of rats #16 and #18). Interestingly, an increase in Tb.Th* was found (+25% at week 12 in relation to baseline p<0.01). Most of these microarchitectural changes were detected at week 2 in the longitudinal study design (BV/TV, Tb.Sp*, Tb.N, Table 1), whereas the cross-sectional study detected them at a later time point (week 4). The OVX + ZOL group showed an initial decrease in Tb.N and increase in Tb.Sp*, similar to the OVX group, as well as an increase in SMI (at week 2 in relation to

5 Osteoporos Int (2010) 21: Table 1 Overview of the structural parameters monitored over time, for each group: comparisons between different time points, within each group Week Ave±SD Ave±SD Ave±SD Ave±SD Ave±SD Sham BV/TV (%) 6.7± ±2.5 (67**) Tb.Th* (μm) 57±3 69±4 (21**,## ) Tb.Sp* μm 306±39 272±32 ( 11**) Tb.N (1/mm) 1.17± ±0.27 (37**) SMI 2.5± ±0.1 ( 6.8**) 13.1±3.3 (96**,#, 18*) 76±4.8 (34**,##, 10**) 277±43 ( 9.2**, 2.1) 1.71±0.36 (46**, 6.8) 2.2±0.1 ( 10**, 3.5) 12.6±4.6 (88**, 3.9) 78±6 (38**,##, 3.4*) 295±54 ( 3.4, 6.4*) 1.58±0.48 (35*, 8.0) 2.3±0.2 ( 6.1, 4.4) 16.4±5.2 (145**,##, 30*) 82±7 (44**,##, 4.6) 260±48 ( 15, 12) 1.99±0.55 (70**,#, 26) 2.1±0.3 ( 13*,#, 7.3) Ovx BV/TV (%) 10.2± ±2.7 ( 27*) Tb.Th* (μm) 62±5 63±3 (2.8) Tb.Sp* (μm) 275±75 339±67 (23**) Tb.N (1/mm) 1.63± ±0.38 ( 29*) SMI 2.3± ±0.2 (6.2) 3.7±1.3 ( 64**,##, 50**) 63±2.6 (1.8, 1.0) 671±233 (144**,#, 98**) 0.58±0.2 ( 64**,#, 49**) 2.6±0.1 (14**,##, 7.8*) 2.4±1.6 ( 76**,##, 35) 71±4 (14*,#, 12**,# ) 1096±272 (299**,##, 63**,## ) 0.34±0.21 ( 79**,##, 43*) 2.6±0.1 (15*,##, 0.3) 2.6±1.2 ( 74*,##, 8.6) 77±5 (25**,##, 9.0) 1058±231 (285**,##, 3.6) 0.35±0.16 ( 79**,##, 3.7) 2.5±0.1 (9.9, 4.3) Ovx + Zol BV/TV (%) 8.6± ±1.4 ( 19) Tb.Th* (μm) 60±5 66±4 (8.5**) Tb.Sp* (μm) 286±36 367±53 (28**,# ) Tb.N (1/mm) 1.40± ±0.17 ( 25*) SMI 2.4± ±0.1 (4.7*) 8.7±2.2 (1.3, 25) 67±3.5 (11*, 2.0) 323±50 (13, 12*) 1.29±0.28 ( 7.8, 23*) 2.4±0.1 (2.8, 1.8) 14.1±3.3 (65*,#, 63*, 104**,## ) 69±4 (14**,#, 3.1, 5.2**) 272±32 ( 4.9, 16, 26**,# ) 2.06±0.52 (47, 60*, 97**,## ) 2.1±0.2 ( 11, 14, 15* # ) 17.6±4.2 (106*,##, 25**, 155**,## ) 74±3 (22**,##, 6.6*, 12**,# ) 255±31 ( 11, 6.4, 31*,## ) 2.41±0.64 (73 #, 17*, 130*,## ) 1.9±0.3 ( 19*,##, 9*, 23**,## ) In parentheses, week 2: (percent difference to week 0); in parentheses, weeks 4, 8, and 12: (percent difference to week 0, percent difference to previous time point); OVX + ZOL group, weeks 8 and 12 in parentheses: (percent difference to week 0, percent difference to previous time point, percent difference to week 2) Ave average, SD standard deviation *p<0.05, longitudinal study design (paired t test); **p<0.01, longitudinal study design (paired t test); # p<0.05, cross-sectional study design (Scheffe's post hoc); ## p<0.01, cross-sectional study design (Scheffe's post hoc)

6 1376 Osteoporos Int (2010) 21: Fig. 2 Plots of the structural parameters (BV/TV, Tb.Th*, Tb.Sp*, Tb.N, and SMI) monitored over time for the three groups of rats examined (SHAM, OVX, and OVX + ZOL groups). In the first column of graphs, at each time point, the average values for each group are given together with the respective standard deviations (error bars). In the second, third, and fourth columns, the graphs are separated by group (SHAM, OVX, and OVX + ZOL groups, respectively), reporting the values of the individual rats. For each group, always the same markers were used in the different plots, to allow a comparison. 2* The treatment with zoledronic acid started at the second week after surgery (OVX + ZOL group)

7 Osteoporos Int (2010) 21: Fig. 3 3D representation of the tibial cancellous bone of three rats (volume of interest 3 mm in height), appertaining to the respective groups (SHAM, OVX, OVX + ZOL), obtained by in vivo micro-ct examination at different time points. The scans were done at baseline (week 0) and repeated on the same rats at weeks 2, 4, 8, and 12 after surgery. In comparison to the normal aging of the SHAM rat, the dramatic bone loss in the OVX rat is visible. The treatment with zoledronic acid started 2 weeks after surgery (OVX + ZOL group), a time point at which the ovariectomized rat showed significant degradation of the structure. In the following weeks, while the OVX rat continued its bone loss, the antiresorptive effects of the zoledronic acid are clearly shown, with full recovery of the bone structure in the OVX + ZOL rat. 2* Initiation of zoledronic acid treatment at week 2 (OVX + ZOL group) baseline; p<0.05, longitudinal study design, Table 1). However, by week 4 (2 weeks after starting the treatment with zoledronic acid), the bony changes in the OVX + ZOL group were reversed, showing an increase in Tb.N and a decrease in Tb.Sp* compared to the previous time point (at week 4 in relation to week 2; p<0.05). In the cross-sectional study design, this reversal was detected at a later time point (at week 8, 6 weeks after treatment start). While by week 4, the OVX group's BV/TV was already dramatically decreased by 64% in relation to baseline, the OVX + ZOL group maintained values similar to baseline at the same time point (2 weeks after treatment start), regardless of the study design. Moreover, in contrast to the OVX group, it was found that at week 8 and continuing until week 12, the BV/TV and Tb.N had a striking increase (+104% and +97%, respectively, at week 8 in relation to week 2; p<0.01), while SMI decreased in both study designs. An increase in Tb.Th* was also found in the OVX + ZOL group in both study designs. At each time point, for each given structural parameter, an additional one-way ANOVA test was done to determine if there were differences in the measured values that were due to treatments. If F values were significant, a Scheffe's post hoc test was done, to compare at that time point the values of the structural parameter between the different groups. At baseline, no significant differences were found between the three groups (Tables 1 and 2, p>0.05). At week 2, significant differences began to be detected, with the two OVX groups having lower BV/TV and Tb.N than the SHAM group. At week 4 (2 weeks after treatment start), BV/TV in the OVX + ZOL group had a value between the OVX and the SHAM group (p<0.05). After this time point, at week 8 and 12, BV/TV in the OVX + ZOL group returned to values of the SHAM group, which were significantly higher than the OVX group (Tables 1 and 2, p<0.001). Similarly, from week 4 until the end, also Tb.N and Tb.Sp* in the OVX + ZOL group returned to values of the SHAM group (Tables 1 and 2). Discussion In this study, a compact in vivo micro-ct scanner was used to monitor the microarchitectural changes in the cancellous bone in three groups of rats over time: a sham-operated group, an ovariectomized group, and an ovariectomized group having zoledronic acid treatment started at 2 weeks after surgery. Measurements were taken within the first 3 months after the surgery, with each group demonstrating different patterns of changes in the cancellous bone microarchitecture over time. For the SHAM group, increases found in BV/TV, Tb.N, and Tb.Th*, together with decrease in SMI, indicates increased bone mass. This is attributed to natural bone growth (Fig. 4), as the animals were aged 2 months at the beginning of the study, with the biggest changes found in week 2 (e.g., +67% in BV/TV). This is in agreement with previous findings on rats, with bone growth reported at 3 months of age [9, 20, 33].

8 1378 Osteoporos Int (2010) 21: Table 2 Statistical significance for the comparison between groups in the average values of the structural parameters reported in Table 1, p values at each time point (Scheffe's post hoc test) Week BV/TV SHAM vs OVX ns <0.05 <0.001 <0.001 <0.001 SHAM vs OVX + ZOL ns <0.05 <0.05 ns ns OVX vs OVX + ZOL ns ns <0.01 <0.001 <0.001 Tb.Th* SHAM vs OVX ns <0.05 <0.001 <0.05 <0.05 SHAM vs OVX + ZOL ns ns <0.01 <0.001 ns OVX vs OVX + ZOL ns ns ns ns ns Tb.Sp* SHAM vs OVX ns ns <0.001 <0.001 <0.001 SHAM vs OVX + ZOL ns <0.05 ns ns ns OVX vs OVX + ZOL ns ns <0.01 <0.001 <0.001 Tb.N SHAM vs OVX ns <0.05 <0.001 <0.001 <0.001 SHAM vs OVX + ZOL ns <0.05 ns ns ns OVX vs OVX + ZOL ns ns <0.01 <0.001 <0.001 ns not significant (p>0.05) SMI SHAM vs OVX ns ns <0.001 <0.05 ns SHAM vs OVX + ZOL ns <0.05 <0.01 ns ns OVX vs OVX + ZOL ns ns <0.01 <0.001 <0.01 The ovariectomized rats showed a general continuous deterioration of the cancellous bone architecture, first detected in the second week after surgery, as found by the longitudinal study design. These changes, a decrease in BV/TV, Tb.N and increase in Tp.Sp* (changed most rapidly between week 4 and 8), are attributed to bone loss. After week 8, the values showed no further significant change, reaching a plateau. These findings of rapid bone loss within the first months after ovariectomy in rats, demonstrated by diminishing BV/TV, Tb. N and increasing Tb.Sp, are in agreement with what has been described in the literature [12, 15, 16, 22, 26, 34]. For example, in a longitudinal in vivo micro-ct study on the same strain of rats [16], the BV/TV measured at week 2 after OVX was decreased by more than 25% and by more than 50% after week 8, which is comparable to our findings (decrease of 27% and 76%, respectively). Similar findings were reported by Brouwers et al. on Wistar rats, with a decrease in BV/TV of 35%, 50%, and more than 50% at week 2, 4, and 8 after ovariectomy, respectively [12]. The effect of zoledronic acid injection was, in the first instance, to prevent the cancellous bone decrease in BV/TV and the deterioration of the microarchitecture due to OVX. At week 4 after ovariectomy, only 2 weeks after treatment start, while the OVX rats had already lost 64% of the bone volume fraction, the OVX + ZOL group BV/TV was similar to baseline. Additionally, while in the first 2 weeks after ovariectomy the values of Tb.Sp* and SMI increased and Tb.N decreased, showing bone loss and deterioration of the cancellous bone structure, 2 weeks of treatment with zoledronic acid completely inverted this trend (Table 1), restoring the baseline values. Therefore, it is shown that the early treatment not only prevented cancellous bone from Fig. 4 SHAM group. Longitudinal cross-section images of the proximal tibia of a rat obtained by in vivo micro-ct imaging at the different time points (weeks 0, 2, 4, 8, and 12), showing natural bone growth. The rat was 2 months old at the beginning of the study (week 0)

9 Osteoporos Int (2010) 21: further deterioration of microarchitecture resulting from OVX, but it restored the structural parameters to baseline. Moreover, at week 8, a striking increase in BV/TV, Tb.N and a decrease in SMI were found, from which point on the parameters exhibited changes resembling the average values of the SHAM group, that is, bone growth, conversed to the OVX group that showed continuous bone loss. In addition, from this time point on, both the OVX + ZOL and the SHAM group differed significantly from the OVX group, thus determining that the overall effect of zoledronic acid in the first 2 weeks of treatment was to preserve and restore the cancellous bone microarchitecture to the values prior to the OVX surgery. In the following weeks, the zoledronic acid treatment resulted in microarchitectural values and patterns of change that were similar to the SHAM group. In general, in the literature, many bone studies are found involving treatments in growing rats [9, 13, 35 39]. The results of those studies, as well as the present ones might be considered by taking into account that the animals are still in their growing phase. It is worth mentioning that in the OVX + ZOL group, an accumulation of a dense layer of cancellous bone close to the growth plate was found (Fig. 5). At week 12, this layer was up to 1-mm thick on average. An observation of this kind was reported in a histological study by Hornby et al. [9] that used zoledronic acid on 3-month-old ovariectomized rats. In that work, the accumulation of a dense layer of cancellous bone beneath the growth plate was visible, reflecting the pharmacodynamic effect of bisphosphonate treatment on longitudinal growth during the initial phase of the experiment. The present study using in vivo micro-ct confirms this histological finding. As described in the Methods section, the volume of interest chosen in the present study started 1 mm distally of the lower end of the growth plate, in the secondary spongiosa as done in previous studies [9, 11, 35, 37]. In a previous in vivo micro-ct study, major changes in the proximal tibia of OVX rats were detected within the first 3 months postovariectomy, suggesting that anticatabolic (antiresorptive) treatment of OVX rats in this early time window may be able to preserve the existing Fig. 5 Longitudinal cross-section images of the proximal rat tibia obtained by in vivo micro-ct at 12 weeks after surgery. From left to right: tibia from the SHAM group, from the OVX group, and from the OVX + ZOL group, respectively. In the OVX + ZOL group, an accumulation of a dense layer of cancellous bone close to the growth plate was observed (black arrow) architecture [11]. In the same work, longitudinal studies with increased frequency of measurements in these first 3 months were also suggested, which should give improved, time-elapsed insight into the early architectural changes. Indeed, in a later study by the same group, major changes were found in the second week, highlighting the importance of the early treatment for bone loss due to the short time frame in which these parameters experience the largest magnitude of change [22]. This suggests the hypothesis of a small window after the initiation of estrogen deficiency in which interventions can be applied to prevent the resulting bone loss, as discussed by Laib et al., Boyd et al. and Campbell et al. [11, 15, 22]. The outcomes of the present in vivo study support this hypothesis, showing the efficacy of beginning antiresorptive treatment within this early period after ovariectomy. The present outcomes of bone maintenance in the proximal tibia over time are comparable with previous findings of zoledronic acid injection given at time of ovariectomy [9, 12], with the difference, however, that here the treatment was started after onset of estrogen deficiency, when significant degenerative changes were already detected. Conversely, there are some substantial differences to the results for late treatment found by a very recent study by Brouwers et al., with zoledronic acid injection given 8 weeks after ovariectomy [12]. In that study, the percentage difference to baseline in bone volume fraction in the proximal tibia decreased from ca. 45% at week 8 (initiation of treatment) towards 35% at week 16, and remained significantly lower than the control values [12]. This corresponds to a partial bone recovery in contrast to a full bone recovery in the present study. This difference in findings is probably due to the later time point at which the zoledronic acid was given in that study (8 weeks compared to 2 weeks). This highlights the importance of the status of the trabecular bone architecture at the time of initiation of the treatment. In the present study, starting treatment 2 weeks after ovariectomy, within the early period in which the structure experiences the major changes, allowed the bone to be fully restored to initial values and maintained to control values over time. This finding might have important implications for future osteoporosis studies, as these first weeks postovariectomy could represent an important time window to effectively recover bone mass. This early time point could be seen analogous to a time window of early intervention in humans, in a first period after onset of menopause [26]. If this is confirmed by future studies, this could have potential clinical implications, based on early initiation of antiresorptive treatment after onset of menopause. Interestingly, in the present work, an increase over time in Tb.Th* was found in all three groups of animals. While this seems reasonable for the SHAM and the OVX + ZOL rats, this is remarkable for the OVX rats. For the OVX rats,

10 1380 Osteoporos Int (2010) 21: a diminishing thickness may be expected over time, which should occur with dramatically decreasing BV/TV [4, 40, 41]. However, varied findings on trabecular thickness in OVX rats are reported in the literature. For example, while some studies report a diminishing thickness in the proximal tibia in OVX over time (e.g., in 8-month-old Wistar rats [11, 22] or 6-month-old Sprague Dawley rats [15]), other studies report no change or an increasing thickness (3-month-old Sprague Dawley rats [9] or 6-month-old Sprague Dawley rats [8], or 8 months Wistar rats [12]). These findings are somewhat controversial and probably require further studies, as the results may also depend on strain of the examined animals [11]. It is also likely that bone loss in OVX rats resulted in actual removal of trabecular elements over time, with preference of removal given to thinner trabeculae, as suggested by the decrease in trabecular number and increase in trabecular separation in the present study [19, 22]. Considering the parameter SMI in OVX rats, a change in the values indicating a transition from a merely plate-like structure to a more rod-like structure is reported in the literature (ideally, SMI values changing from 0 to 3 [11, 15, 22]). Here, the reported average value of 2.3 for SMI at week 0, that increased to a value of 2.6 at week 4 after OVX, follows this trend, indicating a transition towards an almost rod-like structure. However, the actual changes were smaller when compared to some studies reported in the literature. For example, values increasing from SMI=1.2 to SMI=2.8 are reported at 4 weeks after OVX (8-month-old Wistar rats, data read from figure [12]), or changes from SMI=0.9 to SMI=2.6 after 5 weeks (6-month-old Sprague Dawley rats [15]). On the other hand, in another study, SMI increased from 2.1 to 2.4 (10-month-old Wistar rats [19]), which is similar to the increase found in the present study. The secondary aim of the present study was to compare the ability to detect structural bone changes using a longitudinal study design with those of using a crosssectional study approach in a drug treatment study. In a recent in vivo micro-ct study by Boyd et al., the advantage of having each animal acting as its own control in examining ovariectomized and sham-operated rats has been shown [11]. In the present study, the longitudinal study design allowed an earlier detection of the microarchitectural changes including the zoledronic acid-treated group, thus, confirming this advantage also in drug treatment studies. Therefore, it is shown that in a longitudinal study design, the natural variability within a group is less likely to mask changes due to treatment. Another advantage of an in vivo micro-ct examination as opposed to an in-vitro micro-ct examination is the reduced number of animals needed for an adequate, statistically powered study. In the present study, 20 rats divided into three groups were repeatedly examined in vivo at five different time points. Carrying out a similar micro-ct study in vitro, i.e., by using a crosssectional study design, would require at least four times more animals (20+80 rats, total of 100 rats), with 20 rats being euthanased at each time point. As such, the use of a longitudinal study design is beneficial not only for statistical power, but also in issues related to animal ethics, care, welfare, feeding, and housing, as it reduces the number of animals required and cost for these studies. The comparison longitudinal vs cross-sectional design was done by reanalyzing the longitudinal data with a crosssectional approach, similar to the study by Boyd et al. (statistical comparison) [11]. However, it has to be considered that in a real longitudinal vs cross-sectional design comparison, a true cross-sectional study had to be included, which implies that at each time point a different set of animals is examined, with a correspondingly larger total amount of animals, as done in the study by David et al. [20]. That study, which examined a rat model with induced bone loss (tail suspension) and a control rat group over time, demonstrated that bone changes were detected earlier in the longitudinal than in the cross-sectional design, which is the same conclusion on early detection found in the study by Boyd et al., supporting the comparison approach used by Boyd et al. The present study, by using the comparison approach used by Boyd et al., shows earlier detection in a drug treatment study. In conclusion, the present study monitored bone changes in the proximal tibia in sham-operated, ovariectomized, and zoledronic acid-treated rats, using a compact in vivo micro-ct system. Dramatic bone loss was evident within the first weeks after ovariectomy. The zoledronic acid treatment of ovariectomized rats, started 2 weeks after OVX, showed significant effects as early as within the following 2 weeks. It increased the bone mass and fully restored the otherwise deteriorating cancellous bone microarchitecture, bringing it to values similar to the SHAM group within 6 weeks of treatment. This confirms that early antiresorptive treatment facilitates the reversal of both cancellous bone loss and deterioration in the microarchitecture. Furthermore, early detection of the bone changes is important in the study of both pathology and the effectiveness of treatment. In this context, it has been demonstrated that in vivo micro-ct is an invaluable tool for monitoring the bone changes. By applying a longitudinal study design, it provided earlier detection of bone structural changes compared with a cross-sectional study design. Treatment with zoledronic acid as late as 2 weeks after ovariectomy still facilitates the full reversal of cancellous bone loss in rat tibia. Acknowledgments Funding for this work was provided by a grant from the Australian Research Council (DP ). Conflicts of interest Dr. Phil Salmon is an employee at Skyscan NV, Belgium. All the other authors have no conflicts of interest.

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