Noninvasive In Vivo Monitoring of Bone Architecture Alterations inHindlimb-Unloaded Female Rats Using Novel Three-Dimensional

Microcomputed Tomography

VALENTIN DAVID,1 NORBERT LAROCHE,1 BENJAMIN BOUDIGNON,1

MARIE-HELENE LAFAGE-PROUST,1 CHRISTIAN ALEXANDRE,1

PETER RUEGSEGGER,2 and LAURENCE VICO1

ABSTRACT

We tested a novel microcomputed tomograph designed to longitudinally and noninvasively monitor bonealterations in hindlimb-unloaded female rats at a resolution of 26 �m over a period of 3 weeks. This prototypehas a potential to detect three-dimensional trabecular microarchitectural changes induced by growth andunloading.

Introduction: Until now, data concerning structural changes of cancellous bone have only been available afternecropsy of animals. In this study, we tested a novel microcomputed tomography (�CT) technique designed tomonitor such changes repeatedly at a resolution of 26 �m with an acquisition time of about 10 minutes to map theentire proximal tibial metaphysis.Materials and Methods: Four-month-old female Wistar rats were randomized to seven groups of 10 animals to beeither tail-suspended or to act as controls. �CT and DXA measurements were performed at 0, 7, 14, and 23 days insuspended and control rats. One group was killed at each of these time points, and bone samples were processed forhistomorphometry and ex vivo �CT.Results: We verified that a good correlation was obtained between two-dimensional bone parameters evaluated inlongitudinal tibial sections either by histomorphometry or �CT and �CT parameters obtained from either in vivo orex vivo tibias. The longitudinal survey allowed earlier detection of both growth and unloading-related bone changesthan the transverse survey. In controls, aging induced denser bones, reorganization of the trabecular network towarda more oriented plate-like structure, and an isotropic pattern. Unloading first inhibited cortical and cancellous bonegrowth and then induced bone loss characterized by fewer trabeculae, reduced connectivity density, and enhancedstructure model index (SMI), revealing a lighter cancellous structure with development of rod-like characteristics.Conclusion: We show for the first time that this �CT prototype has a great potential to accurately, repeatedly,reliably, and rapidly investigate alterations of three-dimensional trabecular microarchitecture.J Bone Miner Res 2003;18:1622–1631

Key words: true three-dimensional microarchitecture, DXA, bone loss, bone growth, histomorphometry

INTRODUCTION

THERE HAS BEEN a growing interest in microstructuralchanges of cancellous bone, because bone densitometry

cannot entirely account for the observed decrease in bonequality.(1–3) There are current medications that increasebone mass in osteoporotic patients.(4) However, bone

strength could be increased while bone mass is not, sug-gesting that bone quality is the prime factor.(5,6) Palle etal.,(7) in a bed rest study, found that bone microarchitecturewas altered, despite no modification of bone mass.

However, assessment of bone microarchitecture by con-ventional histomorphometry leads to typical problems suchas destruction of the bone sample (embedding and cutting).Moreover, the distribution of cancellous bone is heteroge-neous, and bone histomorphometric analysis is limited to aThe authors have no conflict of interest.

1LBTO, INSERM E0366, University of Saint-Etienne, Saint-Etienne, France.2Institute for Biomedical Engineering, University of Zurich and Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 18, Number 9, 2003© 2003 American Society for Bone and Mineral Research

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few fields of view. The standard procedure leads to evalu-ation of three-dimensional (3D) morphometric indices, de-rived from two-dimensional (2D) images using stereologi-cal methods, and a range of important indices can beindirectly derived from a structural model assumed to befixed. The assumption of a fixed model type is critical,because the structure of trabecular bone may change con-tinuously because of aging and disease effects.

Over recent years, microtomography has become an in-creasingly popular method to measure bone samples, be-cause of its relative rapidity compared with conventionalhistology and its potential as a nondestructive method. Theprogress from 2D to 3D analysis and direct evaluation of 3Dparameters without assuming a fixed model structure havecontributed to the growth of this technique. In rodent bonestudies, higher spatial resolution can be achieved at theexpense of invasiveness, which is the prime factor limitingcurrent computed tomography (CT) scanner developments.The method applied today currently consists of killing theanimal and examining the bone by in vitro microtomogra-phy. The disadvantage of this technique is that the animalcannot be examined at various stages of disease or morethan once during the course of treatment. Other techniqueshave been recently developed to accurately image trabecularbone structure in vivo at a lower resolution.(8) Spatial res-olution and signal/noise ratio are fundamentally limited bythe radiation dose that can be applied: the dose increaseswith the fourth power of linear resolution. For this reason,resolution is generally lower in in vivo measurements, andthe dose becomes an important issue for in vivo measure-ments. Because the bones of rodents, such as rats and mice,are considerably smaller than human bones and because theacceptable radiation dose might be proportionally relativelyhigher than in humans, spatial resolution achieved in labo-ratory animals is clearly better than in humans.

The primary purpose of this study was to evaluate theaccuracy of a newly developed noninvasive experimentalCT scanner. This scanner, a rotating gantry version of thepreviously developed 3D microcomputed tomography (3D�CT), offers high scanning speed and high resolution, at amoderately low radiation dose. This instrument was code-veloped by the Institute for Biomedical Engineering (Insti-tute for Biomedical Engineering, University of Zurich andSwiss Federal Institute of Technology [ETH], Zurich, Swit-zerland) and Scanco Medical (Scanco Medical AG, Bass-ersdorf, Switzerland) and is designed to image small labo-ratory animals in vivo. To validate this tool, wecharacterized the bone changes of tail-suspended rats bysimultaneously assessing bone histomorphometry, in vivoDXA, and in vivo 3D �CT. We compared the bone dynam-ics investigated by these three methods to determine thekinetics of bone loss in this model of osteoporosis.

MATERIALS AND METHODS

Animals

Seventy female rats (Charles River, Iffa Credo,L’arbresle, France), 12 weeks old and weighing 250 � 10 g,were randomly assigned to seven groups of 10 animals each.Rats were tail suspended (S) or were maintained in suspen-

sion cages but not suspended (control groups; Ctr), for 7, 14,and 23 days. A baseline group was killed on the first day ofthe experiment.

The rats were acclimatized for 1 week with standardtemperature (23 � 1°C) and light:dark (12:12) conditions.Animals were individually housed, provided with food(standard diet) and water ad libitum. The suspension pro-cedure was performed according to the Wronski and Morey-Holton(9) recommendations. Fluorochrome double bone la-beling was performed 4 days and 1 day before death. The 7-,14-, and 23-day rats received an intraperitoneal injection of30 mg/kg of tetracycline (Sigma Aldrich). Baseline ratswere not labeled.

�CT and DXA measurements were performed on days 0,7, 14, and 23. Before measurement, rats were anesthetizedby an intraperitoneal dose of 5 mg/kg of ketamine/xylazinesolution, and the animals were killed with a high dose ofNesdonal (Specia, Paris, France).

The left tibia of each animal was measured in vivo and exvivo by �CT and DXA, and bone slices from the samesample were processed for conventional histomorphometry.

Histomorphometric analysis

The proximal tibial metaphysis was fixed in 4% formal-dehyde solution, dehydrated in acetone, and embedded inmethylmethacrylate. Longitudinal frontal slices were cutfrom the embedded bones with the Jung Model K mic-rotome (Carl Zeiss, Heidelberg, Germany). Six nonserialsections, 8 �m thick, were used for modified Goldner stain-ing. Fourteen-micrometer-thick sections were used to deter-mine the dynamic indices of bone formation (double-labeled surface [dLS]/bone surface [BS], mineral appositionrate [MAR], bone formation rate [BFR]/BS). MAR wasderived from fluorochrome interlabel distances. BFR weresubsequently obtained from the product of dLS/BS andMAR. Six-micrometer-thick sections were used for TRACPstaining, allowing determination of osteoclastic parameters.For each section, the data were collected in 2.6-mm-largeregion of interest (ROI) within the secondary spongiosa,according to the areas measured in 2D and 3D �CT (seebelow). Bone volume and parameters reflecting trabecularstructure were measured using an automatic image analysissystem (Biocom). Bone cellular and macroscopic parame-ters were measured with a semiautomatic system:digitizingtablet (Summasketch; Summagraphics, Paris, France) con-nected to a Macintosh personal computer with softwaredesigned in our laboratory.

High resolution �CT

The left tibias of 7-, 14-, and 23-day groups were scannedwith a high resolution �CT prototype (VivaCT20) fromScanco Medical, recently described by Kohlbrenner et al.(10)

(Fig. 1). This apparatus is able to noninvasively examine, invivo, the bones of small laboratory animals (with a diameternot exceeding 20 mm) with a high resolution. The VivaCT20 has a rotating gantry, and the X-ray source and detectorrotate around the object. The spatial resolution of the systemis 20 �m in the x and y axes and 40 �m in the z axis. Thefollowing CT settings were used: voxel matrix 20 � 20 �26 �m3.

1623IN VIVO �CT EVALUATION OF RAT CANCELLOUS BONE

The anesthetized animals were placed inside the Vi-vaCT20, and the left tibia was fixed inside a carbon tube andmeasured. The scanned region corresponded to a zone of253 transverse slices (6.58 mm) under the left proximaltibial growth plate, each slice consisting of 10242 pixels(Fig. 1). The net scanning time was about 10 minutes.

From the acquired data, the ROI in the axial direction wasdelimited anatomically from the bottom of the primaryspongiosa (ISP) up to a height of 2.6 mm (100 slices, Fig.1). Then we adjusted the ROI top at the primary-secondaryfrontier susceptible to vary according to growth or bone lossprocesses. For each transverse slice, the ROI was estab-lished manually in an area of trabecular bone, as large aspossible.

In a preliminary experiment, 10 rats, 5 of which weretail-suspended, were killed and their tibias were processedfor �CT measurements (both in vivo and ex vivo). Thesamples were then processed for conventional histomor-phometry. The scanned acquired images were reprocessedto test the threshold giving the best fit with histomorpho-metric BV/TV data. We therefore defined the optimalthreshold that was used for the rest of the study. All gray-scale images were segmented using a Gaussian filter and afixed threshold (15% of maximal grayscale, correspondingto a value of 150) for all data. The CVs under theseconditions are shown in Table 1.

2D trabecular parameters

Six serial 2D frontal longitudinal slices (104 �m) wereextracted from the acquired images for further evaluation.The axial and transverse ROI (Fig. 1) corresponded tosimilar regions measured by conventional histomorphom-etry: 2.6 mm from the bottom of the ISP and 104 �mlongitudinally (the volume explored by conventional histo-

morphometry was 96 �m). 2D structural indices were cal-culated according to Parfitt et al.(11) for 14 samples selectedat random from all acquired data. Similar areas were mea-sured by histomorphometry and �CT to compare the twomethods.

3D trabecular parameters

The bone volume fraction was calculated directly byplotting gray voxels representing bone fraction againstblack voxels (non-bone objects; VOX BV/TV). Bone vol-ume (BV) and bone surface (BS) were calculated using atetrahedron meshing technique generated by the “marchingcubes method,”(12) and total volume (TV) was calculated asthe volume of the volume of interest (VOI). The normalizedindices (BV/TV, BS/TV, and BS/BV) were used.

Mean trabecular number (Tb.N), mean trabecular thick-ness (Tb.Th), and mean trabecular separation (Tb.Sp) werecalculated according to Parfitt et al.,(11) assuming a constantstructure model and applying stereological techniques.Tb.Th is defined as twice BV/BS, Tb.Sp as 2(TV � BV)/BS, and Tb.N as 0.5BS/TV.

Three-dimensional metric indices were also calculatedusing direct techniques based on the distance transforma-tion,(13,14) without assuming a constant model. Direct indi-ces, indicated by an asterisk to differentiate them frommodel-dependent indices, were calculated as follows:Tb.Th* was calculated by filling maximal spheres into astructure, and the average thickness of all voxels corre-sponded to Tb.Th.

The same procedure was used to determine Tb.Sp, but inthis case the non-bone voxels were filled with maximalspheres, and the mean thickness of the marrow cavitiesrepresented Tb.Sp*.

FIG. 1. VivaCT 20 image acquisition and cancellous bone measurement. A 6.58-mm height zone is acquired (A) during 10 minutes, and then(B) a ROI is chosen that represents (C) a 2.60-mm height zone. (D) Finally the cancellous envelope is extracted from the chosen ROI.

TABLE 1. CV EXPRESSED AS PERCENTAGE AS OBTAINED ON THE DIFFERENT 3D PARAMETERS MEASURED WITH THE VIVACT 20,WHEN MEASURING THE SAME ANIMAL 10 TIMES

Parameters BV/TV Lin.att. Tb.Th.* Tb.Sp.* Tb.N*Conn-dens. SMI DA BS/BV Tb.Th. Tb.Sp.

CV (%) 1.64 0.37 1.94 2.01 1.62 6.17 1.16 0.44 2.30 2.35 1.78

1624 DAVID ET AL.

Tb.N* was the inverse of the mean distance between themidaxes of the observed structure. The midaxes of thestructure were assessed from the binary 3D image using the3D distance transformation and by extracting the centerpoints of non-redundant spheres that fill the structure com-pletely. The mean distance between the midaxes was thendetermined by analogy with the Tb.Sp* calculation, and thedistance between the midaxes was assessed.

The plate-rod characteristic of the structure was estimatedby the structure model index (SMI),(15) calculated by dif-ferential analysis of a triangulated surface of a structure:SMI � 6{[BV(dBS/dr)]/BS2}. dBS/dr is the surface areaderivative with respect to a linear r (one-half thickness orthe radius assumed to be constant over the entire structure).The SMI value is 0 for an ideal plate and 3 for an ideal rodstructure. Values between 0 and 3 correspond to a structurewith both plates and rods, depending on the volume ratiobetween rods and plates.

The geometric degree of anisotropy (D.A.) is defined asthe ratio between the maximal and minimal radius of themean intercept length (MIL) ellipsoid.(16,17)

Connectivity density (Conn.D.) was calculated using theEuler method of Odgaard and Gundersen.(18)

The linear X-ray attenuation coefficient (Lin.Att.), lik-ened to apparent bone mineral density (BMD), was alsoevaluated.

To analyze the cortex, we choose a cross-sectional sliceon the original images for which the distance between thetibia and fibula was approximately 4 mm. We assumed thatthe individual distance was constant, and for each measure-ment point, cortical area (Ct.Ar.), total area (T.Ar.), andmarrow cavity Area (Ma.Ar.) were evaluated, with the sameGaussian filter and the same fixed threshold as for thetrabecular structure.

DXA

A DXA PIXImus densitometer (Lunar Corp., Madison,WI, USA) with small animal software was used to measureBMD and bone mineral content (BMC). It is a rapid (5-minute image acquisition) and precise small animal densi-tometer with a precision of 1% CV for total skeletal BMDand 1.5% CV for femur ROI BMD. After completing themeasurement, the ROI rectangle was moved and reshaped tocover a portion of the left forearm. The PIXI softwareautomatically calculated bone density and recorded the dataas Microsoft Excel files. Entire left tibias and left femorawere analyzed for the 7-, 14-, and 23-day experiments. Lefthumeri were also measured for the 14- and 23-day animals.

Statistical analysis

Statistical analysis was performed using commerciallyavailable statistical software (STATISTICA; StatSoft Inc.,Tulsa, OK, USA). For densitometric and tomographic data,differences between groups were initially analyzed by two-way ANOVA, with a between-groups factor (tail-suspendedor control) and a repeated measures factor (within subjectsfactor). When F values for a given variable were found to besignificant, the sequentially rejecting Bonferroni-Holmtest(19) was subsequently performed using the Holm’s ad-

justed p values. Results were considered to be significantlydifferent at p � 0.05.

Two-way ANOVA was performed on histomorphometricdata to determine the influence of both suspension and timeperiod factors on structural and cellular parameters. When Fvalues for a given variable were found to be significant, apost hoc Scheffe test were performed, and the results wereconsidered to be significantly different at p � 0.05.

Pearson correlation analysis was performed to show po-tential relationships between the various morphometricmethods, and p values were considered to be significant atp � 0.05.

RESULTS

Body weight

A slow but steady increase in body weight was observedin control groups throughout the observation period, asshown in Ctr 23 (Fig. 2). In S 23 rats, an acute decrease inbody weight was observed during the first 3 days followedby a return to baseline values after 1 week. Then S rats grewin parallel to Ctr rats. The body weight in Ctr 7, Ctr 14, S7, and S 14 groups changed in a similar way as in Ctr 23 andS 23 groups (data not shown).

Correlations

Our first aim was to verify that 2D bone parametersevaluated in frontal longitudinal sections either by histo-morphometry or �CT were well correlated. A strong cor-relation was found for BV/TV (r � 0.98; p � 0.0001; Fig.3A), and a significant positive correlation was observed forthe other parameters, such as Tb.N, Tb.Sp, and BS/BV (r �0.781; r � 0.883; r � 0.699, respectively, where p � 0.01).

BV/TV evaluated by histomorphometry was related to 3D�CT BV/TV, although to a lesser extent than in 2D mea-surements, as shown in Fig. 3A (Fig. 3B). Correlationcoefficients between Tb.N, Tb.Sp, and BS/BV measured byhistomorphometry and similar parameters evaluated with orwithout models by 3D �CT ranged between 0.555 and0.732 (p � 0.001). No significant correlation was found forTb.Th.

A significant correlation was also observed between 2Dand 3D parameters measured by VivaCT20 with the samethresholding (0.357 � r � 0.802; p � 0.01).

FIG. 2. Body weight evolution as observed during a 23-day experi-ment. Results are expressed as mean � SD in control animals (filledsquares and solid line) and in suspended animals (open squares anddashed line). (a) Significant difference vs. baseline. *Significant dif-ference vs. Ctr; p � 0.05 ; N � 10 rats per group.

1625IN VIVO �CT EVALUATION OF RAT CANCELLOUS BONE

Significant correlations were observed between all the 3D�CT parameters evaluated in bones scanned either in vivoor ex vivo (excised bones after death); the experiment wascarried out on 20 animals (Table 2).

Histomorphometric assessment of bone alterations

A two-way ANOVA analysis demonstrated that “immo-bilization effects” was the essential factor having a greaterinfluence on architectural modifications than the “growtheffect” factor or the interaction of both factors.

BV/TV and Tb.N increased significantly throughout theexperiment in Ctr rats, revealing a growth effect (Table 3).Bone loss was revealed after the second week of immobi-lization, because BV/TV and Tb.N were significantly lowerin S rats than in Ctr rats. Moreover, a significant increase inTb.Sp was observed in S 23 compared with S 7 rats. Tb.Thremained constant throughout the experiment in both Ctrand S rats, and no significant difference was observedbetween these two groups at any time point. The longitudi-nal growth rate (LGR) did not change significantly, al-

though a trend toward decreases was seen in S 7 and S 14groups compared with the respective Ctr groups.

Dynamic bone formation parameters (BFR/BS) were re-duced in S 14 rats compared with S 7 rats because of adecrease in dLS/BS (Table 4), while MAR remained con-stant throughout the observation period. A group differencebetween S and Ctr rats was observed on day 23. Theseparameters remained unchanged in Ctr rats. Bone resorptionas assessed by Oc.S/BS was not significantly altered by themodel.

Microtomographic evaluation of tibial alteration

Bone growth was accentuated in Ctr rats (Tables 5 and 6),as an increase in bone mass as well as microarchitecturalchanges were demonstrated. Trabeculae became more ori-ented toward isotropic bone (reduction of D.A. on day 14)and organization into plates was observed (reduction in SMIon day 23), reflecting progression of trabecular microarchi-tecture toward a more compact structure. In S rats, a de-crease in BV/TV was observed as early as 7 days (Fig. 4),which further declined with time and was significantlylower than in Ctr rats on day 14. Tb.N decreased signifi-cantly on day 7 in S groups compared with baseline andbecame significantly lower than in Ctr rats on day 14 andremained so until day 23. Tb.Sp increased in S rats on day7 and became significantly higher than in Ctr rats after 14days, and then increased slowly until day 23. Tb.Th re-mained constant throughout the experiment in both Ctr andS rats, and no significant difference was observed betweenthe groups. Variations in 3D metric parameters were fairlysimilar for both model-dependent and model-independentparameters (Table 5). In S rats, the decrease in Lin.Att. from14 days paralleled the decrease in BV/TV. Moreover, adecrease in Conn. Den. was observed from day 7 andsubsequently decreased until day 23. The difference be-tween S rats and Ctr only became statistically significant atday 14. In suspended rats, SMI was more indicative of a rodstructure. This pattern remained stable throughout the ex-perimental period. However, a significant difference wasdemonstrated between S and Ctr rats at days 7 and 14,because SMI was higher in S rats. No significant variationof DA was demonstrated over the same time period. Thissuggests that the growth-related progression toward plateswas inhibited in S rats.

Ct.Ar. significantly increased in Ctr rats over the exper-imental period. Suspension stopped cortical growth (Fig. 5)until day 14, and then induced a decrease of Ct.Ar. NeitherT.Ar. nor Ma.Ar. was significantly altered by aging orsuspension in either S or Ctr rats, although T.Ar decreasedin S rats.

DXA assessment of bone alteration

Left tibia BMD and BMC increased with aging in Ctr ratson day 14. Suspension did not seem to induce any real boneloss in the whole tibia (Table 7), but rather inhibition ofage-related bone growth, because no time-related changes inBMD or BMC were observed in suspended animals. It isnoteworthy that, despite body weight adjustment, baselineBMD or BMC values differed significantly between the

FIG. 3. (A) Correlation between bone volume fraction as measuredby 2D �CT (BV/TV1) vs. volume fraction measured by histomor-phometry (BV/TV). (B) Correlation between bone volume fraction asmeasured by 3D �CT (BV/TV*3D) vs. volume fraction measured byhistomorphometry (BV/TV 2D).

1626 DAVID ET AL.

groups. This was also true for the other 3D �CT boneparameters.

In control animals, femoral BMC increased by 12% after23 days, whereas BMD increased by only 8%. Bone areatherefore increased to a lesser degree than mineral accumu-lation (Table 8). In suspended groups, only an increase ofBMC was observed (3.6% after 1 week, reaching 6.5% at 23days) with no significant variation in BMD. Group differencesfor baseline BMC values were observed, as for the tibia.

Humeral growth was not altered by suspension, becauseBMC and BMD increased at a similar rate in both S and Ctrrats.

DISCUSSION

Our main objective was to determine to what extent 3D�CT parameters acquired in vivo were comparable with

those obtained from optical images of the correspondinghistological sections. For this purpose, frontal longitudinaltomographic sections of the proximal tibial metaphyseswere extracted from the reconstructed excised tibia to matchthe 2D histomorphometric ROI. The 2D �CT BV/TV ratiowas strongly correlated (r � 0.98) with the 2D histomor-phometric BV/TV ratio, despite the lower �CT resolution(nominal resolution 26 �m versus 1 �m for microscopicresolution). Significant correlations were also observed forthe structural parameters (Tb.Th, Tb.N, Tb.Sp) derived fromthe Parfitt-based parallel plate model (r values ranging from0.699 to 0.883), although lower than for BV/TV. Muller etal.,(20) in a study on human iliac crest, also found a highcorrelation between these parameters on histology and �CT(nominal resolution of 14 �m). We also demonstrated thatthe absolute value of BV/TV was similar regardless of the

TABLE 2. CORRELATIONS COEFFICIENTS AND p VALUES BETWEEN THE 3D �CT PARAMETERS EVALUATED IN BONES SCANNED EITHER IN VIVO

OR EX VIVO (EXCISED BONES AFTER DEATH, N � 20)

Parameters BV/TV Linn.att. Tb.Th.* Tb.Sp.* Tb.N.*Conn.den. SMI DA BS/BV Tb.Th. Tb.Sp. Tb.N.

Pearson’ r 0.973 0.972 0.778 0.945 0.959 0.929 0.950 0.402 0.708 0.886 0.975 0.964p �0.0001 �0.0001 �0.0001 �0.0001 �0.0001 �0.0001 �0.0001 �.003 �0.0001 �0.0001 �0.0001 �0.0001

TABLE 3. SUSPENSION AND AGING-INDUCED CHANGES IN LONGITUDINAL GROWTH RATE (L.G.R) AND IN BONE MICROARCHITECTURAL

PARAMETERS IN THE SECONDARY SPONGIOSA OF THE PROXIMAL TIBIAL METAPHYSIS

Group L.G.R. (�m/day) BV/TV (%) Tb.Th. (�m) Tb.N (mm�1) Tb.Sp. (�m)

Basal control Bl Ctr 15.94 � 2.70 46.2 � 2.9 3.45 � 0.44 249.1 � 33.2Ctr 7 27.34 � 2.97 16.76 � 4.36 43.0 � 3.2 3.90 � 0.80 228.2 � 58.7

Controls Ctr 14 24.58 � 3.35 24.98 � 4.13* 40.3 � 7.3 6.35 � 0.78* 121.7 � 16.7*Ctr 23 25.80 � 3.16 23.57 � 2.94* 44.9 � 6.3 5.34 � 0.91* 163.7 � 32.6S 7 26.00 � 3.32 17.01 � 2.90 36.2 � 6.6 4.7130 � 0.20 177.2 � 9.1

Suspended S 14 21.88 � 3.30 18.75 � 2.11‡ 40.3 � 3.9 4.67 � 0.60‡ 177.3 � 24.8S 23 29.16 � 3.25 13.95 � 1.10‡ 40.5 � 3.7 3.61 � 0.55*†‡ 244.9 � 44.6*†‡

Variations in suspended and control animals after 7, 14, and 23 days of experiment.* Significant difference vs. 7 day.† Significant difference vs. 14 day.‡ Significant difference vs. aged matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

TABLE 4. SUSPENSION AND AGING-INDUCED CHANGES IN BONE CELLULAR ACTIVITIES, EVALUATED IN THE SECONDARY SPONGIOSA OF THE

TIBIAL PROXIMAL METAPHYSIS

GroupMAR (�m/

day) dLS/BS (%) BFR (�m/�m2/day) Oc.S/BS (%)

Controls Ctr 7 3.20 � 0.45 8.89 � 1.30 0.28 � 0.06 9.57 � 2.15Ctr 14 3.44 � 0.37 8.30 � 1.42 0.29 � 0.05 8.12 � 1.69Ctr 23 3.07 � 0.40 9.91 � 1.31 0.31 � 0.07 10.04 � 2.42

Suspended S 7 2.56 � 0.60 8.27 � 1.40 0.21 � 0.08 10.65 � 4.89S 14 2.96 � 0.54 4.30 � 2.20*† 0.13 � 0.06* 10.14 � 2.15S 23 2.70 � 0.55 4.87 � 1.47*† 0.13 � 0.08*† 12.97 � 2.98

Variations in suspended and control animals, after 7, 14, and 23 days of experiment.* Significant difference vs. 7 day.† Significant difference vs. aged matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

1627IN VIVO �CT EVALUATION OF RAT CANCELLOUS BONE

technique used. The values of Tb.Sp and Tb.N were fairlysimilar (20% percent difference). However, Tb.Th, as esti-mated by 3D�CT, was 2-fold greater than histological val-ues. An even greater difference has also been previouslyfound in human cancellous bone either by comparing com-puted tomography (150 �m resolution, 330 �m sectionthickness) and histology(21) or by comparing MR-derivedmeasurements of trabecular structures obtained from slicesof the radius (resolution 156 �m, slice thickness 300 �m)with those obtained from high-resolution X-tomographymicroscopy images at an isotropic resolution of 18 �m.(22)

In the present study, no correlation was demonstrated be-tween 2D histomorphometric Tb.Th and 3D �CT Tb.Th(evaluated by either direct or model-dependent methods),although such a correlation was observed for the otherparameters. This lack of correlation does not seem to becaused by insufficient image resolution, which would haveled to significant underestimation of Tb.N, not observed inthis study. It is also not caused by heterogeneity of the sizeof the regions measured (this parameter is very sensitive inmetaphyseal long bone), because ROI were determined verycarefully with the two methods (see Material and Methodssection). In addition to this, we observed that the directTb.Th* measurement was systematically higher than Tb.Thassessed with the plate model assumption by approxima-tively 10%. The reason of this difference might reside in thedeviation of the trabecular structure from the ideal plate-model. In our study, the SMI changed during the experi-mental period in growing rats, but it was globally higherthan 2 (1 corresponds to a plate-like structure and 3 corre-sponds to a rod-like structure), indicating the presence of

more rods than plates. Rods result in a greater surface-to-volume ratio for a given thickness, leading to a smallerapparent thickness derived from BV/BS. Hildebrand etal.(14) found that, even in the human femoral head, whichhas a pronounced plate-like structure, thickness is underes-timated by model-dependent evaluation. Various studies,both in humans(20,21,23) and in the ovariectomized ratmodel,(24,25) have demonstrated the need to use 3D measur-ing techniques that are able to visualize the real architectureof cancellous bone without assumptions concerning the typeof structure. Model-based algorithms may potentially intro-duce biases affecting the parameters determined, and thesebiases may modify the impact of age- or treatment-relatedchanges.

The results of this study show that tail-suspension inducesmetaphyseal bone loss. The longitudinal survey (DXA,�CT) allowed earlier detection of bone changes than thetransverse histomorphometric, tomographic, or densitomet-ric surveys. Initial measurements by DXA and �CT bothrevealed differences in baseline values between the groups,indicating that adjustment based on body weight data didnot ensure homogeneity of bone parameters. Adjustmentshould therefore be performed based on densitometric data.This group heterogeneity might also be detrimental to detectearly changes in a transverse survey. One of the greatestadvantage of carrying out longitudinal studies is to comparechanges against the animal own baseline value.

3D �CT quantification of bone microarchitecture basedon model-independent methods reveals structural modifica-tions earlier than 2D histomorphometric measurements.With a nominal resolution of 20 �m in the x and y axes and

TABLE 5. �CT METRIC PARAMETERS IN THE SECONDARY SPONGIOSA OF THE PROXIMAL TIBIAL METAPHYSIS

Group Period Tb.Th.* (�m) Tb.Sp.* (�m) Tb.N.* (mm�1) Tb.Th. (�m) Tb.Sp. (�m) Tb.N. (mm�1)

ControlsCtr 7 Baseline 76.2 � 10.1 255.2 � 55.7 3.98 � 0.77 67.9 � 12.0 277.5 � 94.3 3.05 � 0.74

7 days 76.8 � 10.4 269.6 � 75.5 3.83 � 0.89 68.1 � 12.2 314.9 � 143.8 2.88 � 0.90Ctr 14 Baseline 90.4 � 14.0 222.2 � 39.8 4.39 � 0.65 82.8 � 15.8 213.5 � 60.4 3.46 � 0.58

7 days 93.1 � 18.3 215.1 � 38.9 4.50 � 0.67 86.0 � 20.4 205.1 � 56.7 3.49 � 0.4814 days 93.1 � 12.8 211.7 � 39.1 4.54 � 0.67 86.8 � 17.7 199.5 � 56.6 3.56 � 0.55

Ctr 23 Baseline 80.6 � 10.7 245.6 � 73.2 4.13 � 0.88 73.1 � 11.5 260.8 � 114.9 3.22 � 0.837 days 82.6 � 12.0 241.2 � 63.1 4.16 � 0.79 74.6 � 13.3 257.3 � 98.4 3.19 � 0.7514 days 86.5 � 11.1 227.8 � 48.8 4.28 � 0.61 78.7 � 13.4 228.9 � 72.3 3.36 � 0.6123 days 88.1 � 12.3 229.8 � 57.7 4.27 � 0.75 81.5 � 13.9 229.6 � 87.2 3.36 � 0.71

SuspendedS 7 Baseline 79.3 � 6.0 252.4 � 47.5 4.00 � 0.63 69.3 � 6.1 277.8 � 71.1 2.98 � 0.58

7 days 80.5 � 6.9 318.4 � 64.3* 3.35 � 0.81* 71.8 � 8.2* 354.9 � 102.6* 2.48 � 0.66*§

S 14 Baseline 90.6 � 12.2 207.8 � 27.3 4.46 � 0.40 82.4 � 11.4 208.2 � 47.8 3.50 � 0.457 days 90.0 � 12.3 236.9 � 39.0* 4.19 � 0.60* 81.5 � 12.5 236.4 � 75.8* 3.26 � 0.63*14 days 86.6 � 13.4* 240.8 � 41.7*§ 4.17 � 0.58*§ 80.0 � 15.0 251.2 � 80.7*†§ 3.13 � 0.61*§

S 23 Baseline 82.4 � 9.2 260.1 � 59.9 3.89 � 0.64 74.5 � 10.7 253.6 � 58.1 3.12 � 0.497 days 84.8 � 8.3 291.0 � 71.8* 3.53 � 0.67* 77.4 � 10.2 286.3 � 78.1* 2.84 � 0.53*14 days 86.2 � 7.3 381.4 � 72.9*†§ 3.02 � 1.01*†§ 79.0 � 7.0 352.2 � 13.6*†§ 2.51 � 0.72*†§

23 days 87.7 � 7.7 410.4 � 77.4*†§ 2.82 � 0.96*†§ 79.9 � 7.8 360.7 � 82.2*†§ 2.52 � 0.63*†§

Bone microarchitectural parameters variations in suspended and control animals, during 7, 14, and 23 days experiment. Animals were measured weekly.Tb.Th*, Tb.Sp*, and Tb.N* represent direct parameters, and Tb.Th., Tb.Sp., and Tb.N represent parameters as calculated using a fixed model assumption.

* Significant difference vs. baseline.† Significant difference vs. 7 day.§ Significant difference vs. aged matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

1628 DAVID ET AL.

26 �m in the z axis, the prototype used for microtomo-graphic acquisitions (VivaCT 20) showed a sufficient dis-criminating capacity to allow precise qualitative and quan-titative study of bone microstructure in vivo.

Because it is difficult and not frequent to use skeletallymature rats (age 12 months or older), we built an experimentwith growing animals. It is necessary to be able to distin-

guish between changes related to inhibition of bone growthand changes attributable to real bone loss. Comparison ofthe kinetics between control and suspended animals andbetween the “baseline control group” (or baseline values forlongitudinal survey) and experimental groups should helpus to make this distinction. No detectable growth effect wasobserved during the first week, but bone loss was alreadydemonstrated after 7 days in the S groups, together with areduction of Tb.N, and an increase in Tb.Sp. Tb.N wasfurther decreased after 7 days. This is also reflected by thedecrease in connectivity density observed after only 7 daysof suspension, which continued to decrease thereafter. Us-

TABLE 6. �CT NON METRIC PARAMETERS IN THE SECONDARY SPONGIOSA OF THE PROXIMAL TIBIAL METAPHYSIS

Group Period SMI DA Conn den (mm�3) Lin att

ControlCtr 7 Baseline 2.50 � 0.65 2.43 � 0.10 35.50 � 16.90 0.932 � 0.083

7 days 2.58 � 0.65 2.38 � 0.07 33.25 � 19.64 0.916 � 0.098Ctr 14 Baseline 2.24 � 0.32 2.32 � 0.08 44.84 � 15.51 1.005 � 0.087

7 days 2.10 � 0.47 2.24 � 0.08 46.74 � 14.11 1.019 � 0.10314 days 1.99 � 0.55 2.20 � 0.08* 48.6933 � 15.756 1.027 � 0.096

Ctr 23 Baseline 2.31 � 0.61 2.35 � 0.07 41.00 � 18.35 0.955 � 0.0957 days 2.30 � 0.66 2.28 � 0.09 40.03 � 16.24 0.961 � 0.09414 days 2.13 � 0.62 2.25 � 0.07* 43.71 � 13.63 0.988 � 0.081*23 days 2.00 � 0.72*† 2.22 � 0.08* 43.35 � 17.25 0.994 � 0.093*†

SuspendedS 7 Baseline 2.63 � 0.34 2.37 � 0.10 34.69 � 12.26 0.928 � 0.057

7 days 2.70 � 0.42 2.31 � 0.11 24.84 � 10.97*§ 0.885 � 0.071*S 14 Baseline 2.06 � 0.50 2.30 � 0.09 45.42 � 10.79 1.011 � 0.060

7 days 2.20 � 0.44§ 2.26 � 0.11 38.92 � 14.51* 0.989 � 0.07914 days 2.28 � 0.52§ 2.27 � 0.11 34.51 � 12.97*†§ 0.976 � 0.080*§

S 23 Baseline 2.28 � 0.50 2.32 � 0.11 38.80 � 10.22 0.944 � 0.0667 days 2.30 � 0.48 2.29 � 0.11 31.55 � 10.20* 0.927 � 0.068§

14 days 2.39 � 0.44§ 2.26 � 0.10 25.90 � 11.13*†§ 0.897 � 0.082*†§

23 days 2.40 � 0.38§ 2.23 � 0.09 23.68 � 9.27*†§ 0.891 � 0.073*†§

Variations in suspended and control animals, during 7, 14, and 23 days of experiment. Animals were measured weekly.* Significant difference vs. baseline.† Significant difference vs. 7 day.§ Significant difference vs. aged-matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

FIG. 4. Bone volume fraction evolution as measured by 3D �CTduring 7 (triangles), 14 (circles), and 23 days (squares). Results areexpressed as mean � SE in control animals (filled objects and solidlines) and in suspended animals (open objects and dashed lines). (a)Significant difference vs. baseline. (b) Significant difference vs. 7 days;*Significant difference vs. aged-matched Ctr; p � 0.05 ; N � 10 ratsper group.

FIG. 5. Cortical area (Ct.Ar.) evolution as measured by �CT duringa 23-day experiment. (a) Significant difference vs. baseline. (b) Sig-nificant difference vs. 7 days. (c) Significant difference vs. 14 days.*Significant difference vs. aged-matched Ctr; p � 0.05. Results areexpressed as mean � SD; N � 8 rats per group.

1629IN VIVO �CT EVALUATION OF RAT CANCELLOUS BONE

ing ex vivo �CT (�CT 20; Scanco Medical AG), we pre-viously reported a pronounced decline in connectivity den-sity in tail-suspended male rats.(26) Complete loss oftrabeculae has been demonstrated in postmenopausalwomen(1) and ovariectomized rats.(24,27) Ito et al.(24) found adramatic decrease in Tb.N and a modest decrease in Tb.Thin female rats after neurectomy. A decrease in Tb.N with asimilar or less pronounced decrease in Tb.Th has beenreported in tail-suspended rats.(26,28) Analysis of bone cel-lular activities in the suspended groups showed thatBFR/BS was unchanged at 7 days, suggesting that thereduction in BV/TV at this time is caused by increased boneresorption, as demonstrated in other models of immobiliza-tion.(29) Trabecular bone loss was accentuated between 7and 14 days of suspension, while the bone formation de-creased during this second week, as shown by the decreasein dLS/BS.

Analysis of 3D �CT nonmetric parameters showed thattail suspension abolishes the tibial growth–induced decreasein both DA and SMI, as seen in control animals. SMI iseven increased by tail suspension. Unloading thereforecounteracts the effects of growth on the bone microarchi-tecture; instead of having a trabecular pattern evolvingtoward a more isotropic and plate-like structure, unloadingpreserves anisotropy and accentuates the rod-like structure.The studies by Ito et al.(24) in neurectomized rats and byLaib et al.(26) in male suspended rats also demonstrated anincrease in SMI. These results emphasize the importance of

assessing real 3D parameters not based on a model assump-tion.

Suppression of growth in the femur shows that the me-tabolism of cortical bone is altered by immobilization, be-cause we observed a decrease in BMC in the S groups,while BMD was not altered. These results suggest that bonearea is altered by tail suspension. Inhibition of femoralgrowth, as observed after 14 days of suspension, is causedby metaphyseal bone loss associated with inhibition ofcortical growth.

In conclusion, we have validated in vivo �CT by com-paring the same 2D parameters measured on histologicalsections and on the same areas reconstructed by tomogra-phy. We then confirmed that the plate-model assumption isnot appropriate to evaluate the kinetics of bone changesduring growth and hindlimb unloading. In our model, thetibial cortical and cancellous bone density and volume in-creased in growing female rats, and their trabecular networkbecome more isotropic and developed a more plate-likeappearance. These effects are counteracted by tail suspen-sion, which initially inhibits the bone growth process andthen induces bone loss accompanied by less numerous andless well-connected trabeculae presenting more rod-likecharacteristics.

We showed for the first time that this �CT prototype hasgreat potential to accurately, repeatedly, reliably, and rap-idly investigate changes in the 3D trabecular microarchitec-ture in a rat model of osteoporosis.

TABLE 7. SUSPENSION AND AGING-INDUCED CHANGES IN BONE

DENSITOMETRIC PARAMETERS OF THE ENTIRE LEFT TIBIAS

Group Period BMD (g/cm2) BMC (g)

ControlsCtr 7 Baseline 0.152 � 0.003 0.290 � 0.007

7 days 0.150 � 0.008 0.293 � 0.017Ctr 14 Baseline 0.147 � 0.008 0.280 � 0.018

7 days 0.146 � 0.010 0.288 � 0.015*14 days 0.152 � 0.007* 0.296 � 0.016*†

Ctr 23 Baseline 0.140 � 0.009 0.276 � 0.0177 days 0.144 � 0.005 0.282 � 0.01514 days 0.148 � 0.012 0.296 � 0.021*23 days 0.151 � 0.011* 0.306 � 0.015*†

SuspendedS 7 Baseline 0.150 � 0.008 0.288 � 0.010

7 days 0.148 � 0.009 0.279 � 0.020S 14 Baseline 0.151 � 0.007 0.304 � 0.018§

7 days 0.155 � 0.007 0.305 � 0.015§

14 days 0.158 � 0.008* 0.309 � 0.022S 23 Baseline 0.147 � 0.011 0.289 � 0.025

7 days 0.147 � 0.010 0.291 � 0.02314 days 0.150 � 0.012 0.290 � 0.02923 days 0.151 � 0.014 0.293 � 0.030

BMD and BMC variations in suspended and control animals, during 7,14, and 23 days of experiment. Animals were measured weekly.

* Significant difference vs. baseline.† Significant difference vs. 7 day.§ Significant difference vs. aged-matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

TABLE 8. SUSPENSION AND AGING-INDUCED CHANGES IN BONE

DENSITOMETRIC PARAMETERS OF THE ENTIRE LEFT FEMORA

Group Period BMD (g/cm2) BMC (g)

ControlsCtr 7 Baseline 0.223 � 0.009 0.393 � 0.015

7 days 0.227 � 0.010 0.438 � 0.023*Ctr 14 Baseline 0.217 � 0.013 0.393 � 0.030

7 days 0.220 � 0.011 0.404 � 0.023*14 days 0.228 � 0.010* 0.419 � 0.023*†

Ctr 23 Baseline 0.210 � 0.011 0.377 � 0.0307 days 0.215 � 0.007 0.388 � 0.024*14 days 0.221 � 0.012* 0.401 � 0.022*†

23 days 0.228 � 0.012*† 0.423 � 0.024*†‡

SuspendedS 7 Baseline 0.219 � 0.011 0.382 � 0.025

7 days 0.218 � 0.010 0.392 � 0.023§

S 14 Baseline 0.220 � 0.010 0.404 � 0.0217 days 0.227 � 0.012 0.419 � 0.026*§

14 days 0.226 � 0.010 0.422 � 0.017*S 23 Baseline 0.219 � 0.014 0.398 � 0.034§

7 days 0.224 � 0.014 0.414 � 0.034*§

14 days 0.222 � 0.017 0.417 � 0.040*23 days 0.224 � 0.020 0.424 � 0.048*

BMD and BMC variations in suspended and control animals, during 7,14, and 23 days of experiment. Animals were measured weekly.

* Significant difference vs. baseline.† Significant difference vs. 7 day.‡ Significant difference vs. 14 day.§ Significant difference vs. aged-matched Ctr; p � 0.05.Results were expressed as mean � SD; N � 10 rats per group.

1630 DAVID ET AL.

ACKNOWLEDGMENTS

We thank Andres Laib, Bruno Koller, and Stephan Haem-merle from Scanco Medical (Scanco Medical AG, Bassers-dorf, Switzerland) for technical support and image analysis.This study was supported by the ERISTO (European Re-search In Space and Terrestrial Osteoporosis) group withthe partners LBBTO, SCANCO, and Institute for Biomed-ical Engineering (contract number 14232/00/NL/SH) andINSERM (Institut National de la Sante et de la RechercheMedicale).

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Address reprint requests to:Laurence Vico, PhD

LBTOEMI 0366

Faculte de Medecine15 rue Ambroise Pare

42023 St Etienne Cedex, FranceE-mail: vico@univ-st-etienne.fr

Received in original form December 10, 2002; in revised formFebruary 19, 2003; accepted March 26, 2003.

1631IN VIVO �CT EVALUATION OF RAT CANCELLOUS BONE