Postpubertal Architectural Developmental Patterns Differ Between theL3 Vertebra and Proximal Tibia in Three Inbred Strains of Mice*

Helen R Buie, Christopher P Moore, and Steven K Boyd

ABSTRACT: An understanding of normal microarchitectural bone development patterns of common murinemodels is needed. Longitudinal, structural, and mineralization trends were evaluated by in vivo �CT over 12time points from 6–48 wk of age at the vertebra and tibia of C3H/HeN, C57BL/6, and BALB/C mice.Longitudinal growth occurred rapidly until 8–10 wk, slowed as the growth plate bridged, and fused at 8–10 mo.Structural augmentation occurred through formation of trabeculae at the growth plate and thickening ofexisting ones. In the vertebrae, BV/TV increased rapidly until 12 wk in all strains. Between 12 and 32 wk, thearchitecture was stable with BV/TV deviating <1.1%, 1.6%, and 3.4% for the C57BL/6, BALB/C, and C3H/HeN mice. In contrast, the tibial architecture changed continuously but more moderately for BV/TV andTbTh compared with the vertebra and with comparable or larger changes for TbN and TbSp. Age-relatedtrabecular deterioration (decreased BV/TV and TbN; increased TbSp and structure model index) was evidentat both sites at 32 wk. In all strains, the cortex continued to develop after trabecular values peaked. Thetemporal plateau of BMD was variable across mouse strains and site, whereas tissue mineral density wasattained at ∼6 mo for all sites and strains. Geometric changes at the tibial diaphysis occurred rapidly until 8–10wk, providing the C57BL/6 mice and C3H/HeN mice with the highest torsional and compressive rigidity,respectively. In summary, key skeletal development milestones were identified, and architectural topology atthe vertebra was found to be more stable than at the tibia.J Bone Miner Res 2008;23:2048–2059. Published online on August 4, 2008; doi: 10.1359/JBMR.080808

Key words: murine, bone architecture, growth, vertebra, tibia

INTRODUCTION

MURINE MODELS HAVE become an important tool for thestudy of genetics and disease because of the ability to

control factors that are not possible in clinical studies. Ani-mals can be inbred to acquire a homogeneous genetic back-ground (i.e., identical twins) with specific phenotypes and avariety of bone characteristics.(1–6) In addition, animal stud-ies allow a high degree of environmental control and offera unique opportunity to systematically study disease(2,4)

and injury.(7,8) The use of these models for bone research,however, requires a detailed understanding and character-ization of normal patterns of development, particularlynearing skeletal maturity. Some important aspects of skel-etal development to consider include longitudinal growth,mineral accumulation, and structural changes.

Bone length in mice is in part genetically determined(1,9);however, it is unclear if variation results exclusively fromdifferences in longitudinal growth rates or also temporaldifferences in growth patterns. It has been established thatgrowth is fastest during the postnatal period and begins toslow at puberty (�: ∼26–31 days; �: ∼28–35 days),(9) after

which males have longer bones than females.(9–11)

Reported growth patterns after puberty are inconsis-tent,(1,10,12,13) even within a given mouse strain, and studieshave primarily focused on C57BL/6J mice. Whereas someof these patterns have been established through cross-sectional study designs, intergroup variability can poseproblems when characterizing temporal developmenttrends. Longitudinal study designs, in which a group of ani-mals is examined repeatedly over time, would complementcurrent knowledge of bone development by establishingtemporal trends.

Mineral accumulation has been primarily assessed bymeasuring BMD, but this is more difficult to interpret com-pared with tissue mineral density (TMD). For example,BMD increases more rapidly during puberty comparedwith the postnatal period(9) but may reflect slowing growthrates rather than increased mineral deposition becauseBMD depends on both mineral content and total bone vol-ume (i.e., volume of bone and marrow). TMD on the otherhand, which depends on mineral content and tissue bonevolume (i.e., volume of bone only), was found to increasemore rapidly during the postnatal period.(13) Large differ-ences in BMD between two mice strains that have similarmineral size, shape, and organization within the collagenmatrix was found to result from variations in tissue bonevolume,(14) further highlighting the difficulties in relating

*Part of the data herein was presented as poster 938 at the Or-thopaedic Research Society Annual Meeting, San Francisco, CA,USA, March 2–5, 2008.

The authors state that they have no conflicts of interest.

Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 23, Number 12, 2008Published online on August 4, 2008; doi: 10.1359/JBMR.080808© 2008 American Society for Bone and Mineral Research

2048

JO802108 2048 2059 December

BMD to mineral accumulation. TMD provides a more di-rect understanding of mineralization but has rarely beenreported. Recently, bone mineralization was shown to pro-ceed rapidly in postnatal BALB/cByJ mice, with TMDreaching 51% of mature values at 1 day of age and 80% by40 days.(13) Variation in TMD across anatomic sites has notbeen examined, but BMD is clearly site specific.(1,15) De-spite these limitations, timing of peak BMD measured byBeamer et al.(1) is commonly adopted as an indicator ofskeletal maturity.(2,3,10,16–19)

Developmental patterns of bone structure have primarilyfocused on the cortex(9,10,12) or the trabecular compartmentof C57BL/6J mice.(11,20) Architectural parameters havebeen found to peak early and at different times for thecortical and trabecular compartments.(11) Similar develop-mental patterns were found for the tibia, femur, and verte-bra, with more pronounced changes in the long bones.(11)

No stable period of architecture was identified in the ver-tebra, tibia, or femur, and loss occurred rapidly as of 2 moof age, with more marked changes in architecture for fe-males versus males.(11,20) In these studies, few data pointswere acquired around skeletal maturity, even though this isa critical period of development. Further study is needed ofthis period for C57BL/6 mice and for other commonly usedstrains, such as C3H/HeN and BALB/C.

Although substantial data exist in the literature on bonedevelopment, the information is often difficult to interpretbecause of sparsely available data. Therefore, the objectiveof this study was to identify the temporal occurrence of keydevelopmental stages in vivo (e.g., longitudinal growth,mineral accumulation, and structural changes) and themechanisms of architectural augmentation during develop-ment of the vertebra and tibia in three common inbredstrains of mice.

MATERIALS AND METHODS

Mice

Female C3H/HeN (C3H), C57BL/6 (B6), and BALB/c(BAL) inbred mice were acquired at 4 wk of age fromCharles River Laboratories (Quebec, Canada). The micewere maintained in an accredited animal care facility atroom temperature under a 12-h light/dark cycle and givensterile PICO-VAC Laboratory Mouse Diet 5062 (PurinaMills, St Louis, MO, USA) and filtered water ad libitum.The mice were assigned to weight-matched groups for high-frequency and low-frequency scanning (n � 5–6 per groupper strain).

µCT

�CT scanning was performed in vivo (vivaCT 40; ScancoMedical AG) at 12 time points from 6 to 48 wk of age forthe high-frequency (high) scanning group, with higher sam-pling at earlier time points. The low-frequency (low) groupwas sampled at every second time point starting at 8 wk ofage for the B6/C3H and 10 wk for the BAL, yielding sixscans in total to control for the possible effect of radiation.The BAL mice were not scanned at 8 wk because previousdata suggested these mice would mature later than the

other strains.(1) Mice were sedated with isoflurane anes-thetic and maintained on low doses for the duration of themeasurements (∼40 min per mouse). An ophthalmic oint-ment (BNP Sterile Ophthalmic Ointment; Vetcom) was ap-plied to each eye to prevent dryness and damage. Using anovel whole body scanning device (Scanco Medical AG),scans were acquired (55 kV, 109 �A, 200-ms integrationtime, 38.9-mm field of view, 2000 projections on 360°, 2048CCD detector array, cone-beam reconstruction) of thethird lumbar (L3) vertebra and both proximal tibias at anominal isotropic resolution of 19 �m. The L3 vertebra wasselected for evaluation over other larger lumbar vertebraebecause it fit in one stack of �CT images. The deliveredradiation dose was ∼188 mGy based on previous measure-ments in the laboratory using an ionizing chamber probe.This dose is approximately four times lower compared withother in vivo studies.(21) The scanner was calibrated weeklyusing hydroxyapatite (HA) phantoms. After each scan, themice were weighed (Ohaus Scout SC2020), and body masswas recorded. After the final measurement, the mice werekilled by CO2 asphyxiation. The protocol for this study wasapproved by the Animal Care and Use Committee at theUniversity of Calgary.

Image processing

Volumes extracted for analysis included the metaphysisof the vertebral body, the left and right proximal tibial me-taphyses (a 1.5-mm slab extending away from the growthplate), and the left tibial diaphysis (a 0.5-mm slab beginning5.0 mm distal to and extending away from the proximalgrowth plate). Semiautomated hand-drawn contours wereused to isolate regions for analysis, and an automated ap-proach(22) was used to separate the cortical and trabecularcompartments. For the tibia, the long axis of the bone wasfirst aligned with the superior–inferior axis before definingthe different regions of interest. These regions of interestwere then transformed to allow the analysis to be carriedout in the bone’s original scan orientation. Finally, all gray-scale images were processed using a Gaussian filter (sigma� 0.7, support � 1) to reduce noise and a global threshold(22% of the maximal grayscale value) to extract the bonefrom surrounding soft tissue.

Analysis

Bone architecture was assessed by direct 3D meth-ods(23–25) (Image Processing Language v. 5.01a; Scanco),densitometry from the X-ray attenuation, strength indica-tors from cross-sectional geometry,(26) and longitudinalgrowth from vertebral body length. Temporal longitudinalgrowth patterns should not depend on the site examinedbecause relative growth rates are comparable acrosssites.(15) Architectural measurements for the vertebral bodyand the tibial metaphysis included bone volume ratio (BV/TV, %), trabecular thickness (TbTh, �m), trabecular sepa-ration (TbSp, �m), trabecular number (TbN, mm−1), struc-ture model index (SMI), degree anisotropy (DA),connectivity density (ConnD, mm-3), and cortical thickness(CtTh, �m). Mineral accumulation was assessed by densio-metric measures of BMD (mg HA/cm3) and TMD (mg

MURINE SKELETAL GROWTH AND ARCHITECTURAL STABILITY 2049

HA/cm3) for the whole L3 vertebra and for the proximaltibial metaphysis. BMD and TMD are determined by nor-malizing mineral content from the X-ray attenuation bybone volume (bone and marrow) and tissue volume (boneonly), respectively. Relative TMD was also calculated as apercentage of plateau values (TMD, %). Cortical area(CtA, mm2) and polar moment of inertia (J, mm4) of thetibial diaphysis were measured to provide indications of thebone’s ability to withstand axial and torsional loading, re-spectively.(26) In addition, total cross-sectional area (TA,mm2) and medullary area (MA, mm2) provided indicationsof change to the periosteal and endocortical surfaces.

Standard descriptive statistics were evaluated for all vari-ables. To check if the high- and low-frequency scanninggroups could be pooled, architectural outcomes were testedby a two-way ANOVA for mouse strain and treatment(high versus low dose). All continuous dependent variableswere averaged across time, and one- and two-way repeated-measures ANOVAs were used to determine effects ofstrain, site, and limb. Simple effects testing was performedwhere appropriate. Temporal trends were evaluated by aone-way repeated-measures ANOVA excluding incom-plete datasets. Statistical analyses were performed withSPSS 16.0 for Windows (SPSS, Chicago, IL, USA). Differ-ences were considered statistically significant for p < 0.05.

RESULTS

There were no significant differences in the variablesstudied between the high- and low-frequency groups (Fig.1); therefore, the groups were pooled to increase statisticalpower.

The total number of datasets available for analysis wasreduced because of accidental death, disease, operator er-ror, and motion artifacts. Three C3H mice were found deadat 44 wk after a water bottle flooded the cage. Five B6 mice(three high-frequency group; two low-frequency group) de-veloped ulcerative dermatitis, which did not improve withtopical antibiotic treatment (Topagen Spray; ScheringCanada) and were killed at 25, 29, 29, 32, and 37 wk. FemaleB6 mice are prone to this disease and may be more suscep-tible during the summer months,(27) which is when we sawdisease onset. A gross necropsy showed no other abnor-malities. Because of the reduced number of animals, all B6mice followed the high-frequency scanning schedule as of

32 wk. Data are not included for diseased mice. Because ofan operator error, one C3H mouse fell from the scanningdevice at week 12, and data from the left tibia was excludedfrom week 14 onward because it did not follow normaldevelopment patterns. In summary, of the 857 data sets thatwere acquired for normal healthy mice, 19 were discountedbecause of motion artifacts (vertebra: 3 C3H, 3 B6; tibia: 7C3H, 4 B6, 2 BAL). Of the initial 33 mice, a total of 25survived to 48 wk of age (C3H: 4 high, 4 low; B6: 3 high, 3low; BAL: 6 high, 5 low).

Body mass

Body mass increased rapidly until 6 wk of age and thencontinued linearly at a slow and steady rate (Fig. 2A). Onaverage, mass was not significantly different between theC3H and B6 mice, and both were heavier than the BALmice (p < 0.001) because of more rapid increases after week6. Body mass continued to increase for the B6 mice through48 wk, whereas it stabilized in the C3H and BAL mice atweeks 32 and 42, respectively.

Longitudinal growth

Longitudinal growth patterns were strain dependent andproceeded rapidly until 8–10 wk, followed by a slow con-tinuous growth until fusion of the growth plate (Fig. 2A).Longitudinal growth has previously been shown to be mostrapid during the postnatal period and slows at puberty.(9)

We found that growth slows further at 8–10 wk because ofbridging of the growth plate, which we observed at the tibia.There was insufficient resolution to detect bridging ele-ments at the vertebra in this study but is apparent in pre-vious studies,(11) with similar timing to the tibia. Completefusion of the growth plate occurred between 8 and 10 mo.Growth curves were not significantly different between theB6 and BAL mice. C3H mice, on the other hand, had alonger rapid growth period and grew 40% faster between 6and 8 wk, leading to significantly longer bones (p < 0.001).

Mineralization

Mineral accumulation was similar for all three strains butwas only evident from TMD measurements (Fig. 2B). BMDmeasurements were highly variable between anatomic siteand strain with peak or plateau values attained between 14and 34 wk. Plateau values for TMD on the other hand

FIG. 1. Representative lackof variation in architecture be-tween the high- and low-frequency scanning groups.Error bars represent SD.

BUIE ET AL.2050

occurred at ∼26 wk for all sites and strains. A completeTMD (%) curve was obtained by including data fromMiller et al.(13) and can be described by the general growthcurve (r2 � 0.954) given in Eq. 1.

TMD �%� =

21.06 +82.81

1 + 2.31 � exp�−0.77�Agein weeks − 0.08�0.43�[Eq. 1]

Structure

Temporal changes in architectural parameters are plot-ted in Figs. 3 and 4, and representative 3D structures areshown in Figs. 5 and 6. Strain-related differences in archi-tecture were significant at both the vertebra and tibia andsite-to-site differences for the B6 and BAL mice (Table 1).

On maturity, the vertebral body was architecturallystable, with some trabeculae at 6 wk persisting throughweek 48 (Fig. 5). Vertebral BV/TV increased by 113–159%between 6 and 12 wk, primarily because of increases inTbTh, which ranged from 60% to 84% during this period.Between 12 and 32 wk, the mean BV/TV deviated <1.1%,1.6%, and 3.4% in the B6, BAL, and C3H mice, respec-tively. During this period, changes in TbTh, TbN, TbSp,SMI, and DA were minimal, but significant reductions inConnD occurred for the B6 and the C3H mice. Corticalmaturity was delayed compared with the trabecular com-partment, with CtTh attaining maximum values at 17 wk inthe B6 and C3H mice and 25 wk in the BAL strain. Typicalchanges with advanced age (decreased BV/TV and TbN;increased TbSp and SMI) onset at 32–34 wk, but decreasing

BV/TV trends only reached significance for the B6 andC3H strains (B6, p � 0.003; C3H, p � 0.008; BAL, p �0.211).

In contrast to the stability of the vertebra, there werecontinuous changes in architecture at the tibia with age(Figs. 3 and 4). Initial increases in BV/TV and TbTh weremore gradual (less pronounced) than in the vertebra at33–60% and 9–25%, respectively. In contrast, trends inTbN and TbSp were similar to the vertebra but with com-parable or larger changes. There was an early decline inConnD at both sites. The temporal plateau of BV/TV at thetibia differed among mouse strain and did not coincide withthose of the vertebra. However, timing of peak CtTh in theB6 and BAL strains was similar for the vertebra and tibia.As with the vertebra, age-related bone loss was seen at32–34 wk (decreasing BV/TV trends: B6, p � 0.005; C3H,p � 0.009; BAL, p � 0.023).

Developmental trends for the left and right tibias werecomparable, but there were significant differences in tra-becular architecture. For all strains, architectural param-eters were on average higher (p < 0.001) in the right limbcompared with the left, with larger differences between 12and 26 wk. For all the mice, cortical thickness was not sig-nificantly different between limbs at any time point.

Temporal changes in cross-sectional geometry of thetibial diaphysis are provided with representative sections inFig. 7. Data from Richman et al.(9) are included for databefore week 6, and strain-related differences in structureare provided in Table 2. Initial increases in cortical areafrom week 6 arose primarily from apposition to the endo-cortical surface, which occurred rapidly in the C3H and B6mice until 8–10 wk and more moderately until week 20 in

F I G . 2 . ( A ) T e m p o r a lchanges in body mass and ver-tebral body height along withimages showing progressivebridging of the tibial growthplate. (B) Mineralization datafor all strains and sites as as-sessed by BMD and TMD.Data are included from Milleret al.(13) Error bars representSD.

MURINE SKELETAL GROWTH AND ARCHITECTURAL STABILITY 2051

the BAL mice. The B6 mice developed bones with largertotal cross-sectional areas compared with the other strains,leading to enhanced torsional resistance, whereas the C3Hmice developed high cortical area providing superior com-pressive resistance. Structural increases in compressive andtorsional rigidity occurred early in the B6 and C3H miceand more gradually in the BAL mice.

DISCUSSION

Longitudinal, structural, and mineral developmental pat-terns were determined from an in vivo study providing adetailed understanding of normal growth patterns at skel-etal maturity in B6, BAL, and C3H mice at the vertebraand tibia. Despite common use of these mice, skeletal de-

velopment of the BAL and C3H strains has not been wellcharacterized. Whereas the B6 mice have been examinedpreviously,(10,13) this study yields a comprehensive descrip-tion of key developmental stages at maturity. Establish-ment of normal development patterns is not only necessaryto effectively plan studies but can also aid in interpretationof existing ones. For example, our laboratory recently per-formed an ovariectomy or sham operation on these threestrains of mice at 12 wk of age and examined bone loss atthe tibia in vivo over a 5-wk period. Whereas continuousdecreases in BV/TV occurred for the B6 and C3H mice, forunknown reasons, the BAL showed some recovery by theend of the study. This recovery, however, can be explainedby knowing that BV/TV is increasing during this period forthe BAL mice but not for the B6 and C3H. Knowledge of

FIG. 3. Temporal changes incommonly reported architec-ture parameters in the high-frequency scanning group forthe C57BL/6 (left column),BALB/C (center column), andC3H/HeN (right column) micefor the vertebra (solid datapoints) and the proximal tibialmetaphysis (open data points).For the tibia, the average is re-ported for the left and rightlimbs. Because of animal eu-thanasia, the C57BL/6 groupswere combined at 32 wk. Errorbars represent SD.

BUIE ET AL.2052

normal bone changes can also be important for studies pro-viding only endpoint data, which is common in exercisestudies of rodents.(28,29) In these studies, it is difficult toknow if exercise induced positive bone changes or simplyprevented deterioration.

The longitudinal analysis reinforces our current under-standing of bone development by clarifying the temporalchanges in architecture. Structural augmentation occurredin all three strains of mice through formation of new tra-beculae at the growth plate and thickening of existing ones.All three strains of mice exhibited a period of high archi-tectural stability in the vertebra, whereas continuouschanges occurred in the tibia. This developmental differ-ence most likely resulted from the slow growth period whilethe growth plate was bridging, which resulted in replace-ment of a large proportion of the metaphyseal region for

the tibia compared with the vertebra. Despite these struc-tural differences, tissue mineralization patterns (TMD)were similar across both sites and all three mouse strains.Longitudinal growth patterns differed in both rate and keydevelopmental milestones with strain-related differences inbone length primarily established during the rapid growthperiod.

A limitation of this study is that earlier time points maybe underestimated because of partial volume effects andundermineralization (75–100% of mature values) of thebone. However, higher-resolution in vivo scans were notpossible, and multiple thresholds to account for variationsin mineralization could not be selected objectively or con-sistently through manual or iterative methods. Nonetheless,this limitation is likely not too severe because, for the B6mice, the patterns we found were generally consistent with

FIG. 4. Temporal changes inless commonly reported archi-tecture parameters in the high-frequency scanning group forthe C57BL/6 (left column),BALB/C (center column), andC3H/HeN (right column) micefor the vertebra (solid datapoints) and the proximal tibialmetaphysis (open data points).For the tibia, the average is re-ported for the left and rightlimbs. Because of animal eu-thanasia, the C57BL/6 groupswere combined at 32 wk. Errorbars represent SD.

MURINE SKELETAL GROWTH AND ARCHITECTURAL STABILITY 2053

those of cross-sectional studies of C57BL/6J mice, whichhave a similar origin but are from a different supplier.(11,20)

Also, the source, seasonality, and litter size may be factorsthat could result in small differences in the developmentpatterns reported here; therefore, these data should not beconsidered a replacement for the inclusion of normal con-trol experimental groups in future research.

Radiation effects are a consideration with in vivo scan-ning. In this study, the high and low radiation groups pro-duced the same architectural trends from 6 to 48 wk of agedespite being subjected to different radiation doses. Previ-ous reported effects of radiation are variable and rangefrom none in mature rats(21,30) to detectable in growingmice.(21) In the latter study, radiating one limb four timeswas found to result in decreases of BV/TV and TbN andincreases of TbSp compared with the contralateral limb, butdoses were almost 4-fold higher than in this study (712 ver-sus 188 mGy), and the scanning interval was much shorter(1–2 versus 2–12 wk).(21) In this study, radiation effects areunlikely because differences between the high and low ra-diation groups were not significant, and the high radiationgroup did not consistently have lower BV/TV and TbN orhigher TbSp than the low radiation group.

Longitudinal growth patterns in the mouse are consistent

with other reports and clarify some previous inconsisten-cies.(1,10,13) Longitudinal growth of the vertebra proceededrapidly until ∼8–10 wk, followed by a slow continuousgrowth as the epiphyseal plate became bridged and fullyfused at ∼8–10 mo. Previous reports also describe rapid andslow growth periods with either no further growth after 6mo(10,13) or continuous growth through 12 mo.(1) These dis-crepancies can be attributed to less frequent sampling andintergroup variability. Reports of growth patterns in the ratare similar to those found here, with comparable timing forthe rapid to slow transition(31) and fusion of the growthplate.(32) Longitudinal growth rates and timing were similarbetween the BAL and B6 strains, whereas they were higherand extended in the C3H mice, leading to notably largermice at the end of the rapid phase. Both rate and temporaldifferences in growth patterns during the rapid growthphase were important in establishing large differences inbone length between strains.

To our knowledge, this is the first study to examine tem-poral changes to the vertebral structure in vivo with suffi-cient resolution to examine individual trabeculae. Growthclearly followed the model of endochondral ossification andgrowth of long bones,(33,34) with new trabeculae formingonly at the growth plates. High architectural stability was

FIG. 5. Longitudinal changes in the trabecular architecture of the L3 vertebral body from 6 to 48 wk of age from a single representativeanimal of the C3H/HeN, C57BL/6, and BALB/C mouse strains. Images are shown for only 9 of the 12 time points collected. Thearchitecture was most stable between 12 and 32 wk. Typical changes with advanced age onset as of week 32 but were only significantfor the C3H/HeN and C57BL/6 strains. Similar to in humans, bone loss is most prominent in the center of the vertebra.(60)

BUIE ET AL.2054

found after maturation, which is consistent with studies ofrats that have found that minimal modeling occurs in thevertebra from 8 wk onward.(35,36) In a previous cross-sectional study, rapid structural declines were reported infemale B6 mice immediately after peak values werereached at 2 mo.(11) Some possibilities for the discrepancymay be differences in site (L3 versus L5), region of analysis(distance to the growth plate), supplier (Charles River ver-sus Jackson), body composition, and processing methods(constant global versus more sophisticated thresholding).Interestingly, the structural trends for the male B6 mice(11)

were very similar to those found here.In contrast to the high stability of the vertebral structure,

the structure was continuously changing in the proximaltibial metaphysis. Structural development patterns for thetibia were consistent with previous findings for long

bones.(11,20) The higher variability at this site may arisefrom greater degrees of modeling because of continued lon-gitudinal growth.(36) Significant differences in trabecularbut not cortical properties were found between left andright limbs at maturity (data not shown). Asymmetry inbone properties has been reported in humans(37–44) androdents.(30,45) The use of the contralateral limb as an inter-nal control, which has become common in bone stud-ies,(38,46–51) should therefore be used with discretion (e.g.,randomize left and right limbs).

The onset of age-related bone declines we observe issimilar in timing to the progressive lengthening of estruscycles observed in virgin C57BL/6J mice by 9 mo of age.(52)

In a clinical study, BMD was shown to decline in perimeno-pausal woman with increased cycle length and irregularity(oligomenorrhea) but not those with regular cycles (eume-

FIG. 6. Longitudinal changes in the trabecular architecture of the proximal tibial metaphysis from 6 to 48 wk of age from a singlerepresentative animal of the C3H/HeN, C57BL/6, and BALB/C mouse strains. Images are shown for only 9 of the 12 time pointscollected. Continuous changes to the architecture are apparent. Arrowheads are used to show movement of some trabeculae andprovide an indication of replacement of the trabecular region with continued slow growth. Typical changes with advanced age onset asof week 32 for all three strains.

MURINE SKELETAL GROWTH AND ARCHITECTURAL STABILITY 2055

horrhea).(53) Cycle irregularity was associated with de-creased estradiol and increased follicle-stimulating hor-mone,(53) changes that favor bone resorption.(54–56) Theage-related declines we observed may therefore result fromreduced ovarian function preceding ovarian failure.

Patterns of bone loss in the mouse differed somewhatfrom human patterns of bone loss, consistent with long-term studies of C57BL/6J mice.(11,20) Characteristic age-related changes in humans include reduced BV/TV, TbN,and TbTh and increased TbSp.(57–59) Preferential loss of

horizontal trabeculae leads to increased DA,(60) whereasthinning leads to more rod-like trabeculae reflected by in-creased SMI.(61) For both the tibia and vertebra, we foundthat bone loss in mice resulted primarily from decreasedtrabecular number as opposed to thinning. The lack ofchange in DA over time suggests there is no preferentialloss of horizontal trabeculae in mice. However, similar tohumans, we observed that bone loss in the vertebra of themice was more prominent in the vascular region (i.e., centerof the vertebra).(60) The period of bone loss was relatively

TABLE 1. TWO-WAY ANOVA OF ARCHITECTURAL OUTCOMES, AS MEASURED FROM �CT AND AVERAGED FROM 6 TO 48 WK BY

SITE (L3, LUMBAR SPINE, PROXIMAL TIBIA [PT])

Outcome SiteC3H/HeN

(mean ± SE)BALB/C

(mean ± SE)C57BL/6

(mean ± SE) Site F (df) [p] Strain F (df) [p] Site × strain F (df) [p]

BV/TV (%) L3 26.4 ± 0.75*† 5.1 ± 0.86‡§ 33.9 ± 0.83‡§ 671.3 (1,30) [<0.001] 15.5 (2,30) [<0.001] 230.4 (2,30) [<0.001]PT 26.0 ± 1.0*† 21.6 ± 0.7†‡§ 8.8 ± 0.3*‡§

TbTh (�m) L3 98 ± 2† 98 ± 2†§ 89 ± 2*‡§ 152.9 (1,30) [<0.001] 26.9 (2,30) [<0.001] 45.3 (2,30) [<0.001]PT 97 ± 2*† 86 ± 1†‡§ 73 ± 1*‡§

TbSp (�m) L3 347 ± 5*† 247 ± 2†‡§ 209 ± 3*‡§ 234.6 (1,30) [<0.001] 21.4 (2,30) [<0.001] 126.5 (2,30) [<0.001]PT 359 ± 14† 347 ± 11†§ 602 ± 18*§

TbN (mm−1) L3 3.33 ± 0.05*† 4.51 ± 0.04†‡§ 5.10 ± 0.06*‡§ 608.2 (1,30) [<0.001] 11.8 (2,30) [<0.001] 272.1 (2,30) [<0.001]PT 3.32 ± 0.11† 3.22 ± 0.08†§ 1.83 ± 0.05*‡§

ConnD(mm−3)

L3 67.5 ± 2.4*† 76.7 ± 2.3†‡§ 106.1 ± 4.5*‡§ 488.0 (1,30) [<0.001] 1.38 (2,30) [0.267] 216.4 (2,30) [<0.001]PT 67.0 ± 4.1*† 46.3 ± 2.5†‡§ 19.5 ± 2.1*‡§

SMI L3 1.00 ± 0.09*†§ 0.31 ± 0.10†‡§ 0.80 ± 0.10*‡§ 790.4 (1,30) [<0.001] 917.7 (2,30) [<0.001] 110.4 (2,30) [<0.001]PT 1.37 ± 0.07*†§ 1.63 ± 0.06†‡§ 2.67 ± 0.04*‡§

DA L3 1.31 ± 0.01*†§ 1.46 ± 0.01‡ 1.43 ± 0.02‡§ 115.3 (1,30) [<0.001] 39.4 (2,30) [<0.001] 23.3 (2,30) [<0.001]PT 1.44 ± 0.01†§ 1.49 ± 0.01† 1.64 ± 0.02*‡§

CtTh (�m) L3 216 ± 4*†§ 175 ± 3†‡§ 162 ± 3*‡§ 879.1 (1,30) [<0.001] 105.6 (2,30) [<0.001] 30.1 (2,30) [<0.001]PT 249 ± 2*†§ 234 ± 2†‡§ 194 ± 2*‡§

Significantly different (p < 0.05) than *BALB/C, †C57BL/6, and ‡C3H/HeN within the site.§ Significantly different (p < 0.05) between L3 and PT sites within the strain.BV/TV, bone volume ratio; TbTh, trabecular thickness; TbSp, trabecular separation; TbN, trabecular number; ConnD, connectivity density; SMI,

structure model index; DA, degree anisotropy; CtTh, cortical thickness.

FIG. 7. Temporal changes intotal area (TA), medullaryarea (MA), cortical area (CtA),and polar moment of inertia (J)at the tibial diaphysis, 5 mmdistal to the proximal growthplate along with representative2D images. Changes to theperiosteal and endocorticalsurfaces can be inferred fromTA and MA and compressiveand torsional rigidity from CtAand J. Normalized data are in-cluded from Richman et al.(9)

Error bars represent SD.

BUIE ET AL.2056

short in this study; for long-term changes, the reader mayconsult Halloran et al.(20) or Glatt et al.(11)

The vertebra and tibia shared some similarities in devel-opment despite differences in structural stability. Trendsfor TbN and TbSp were similar across sites but with com-parable or larger changes in the tibia. For the B6 and C3Hmice, structural inhomogeneity increased with age at bothsites because trabeculae were not formed and resorbed con-sistently (TbN was time dependent). In addition, age-related changes onset with similar timing at both sites.

In all mice, the mechanisms of architectural augmenta-tion were (1) formation of new trabeculae at the growthplates and (2) thickening of existing trabeculae. The abilityto form new trabecular connections away from the growthplate has been a contentious issue(62) because of the re-duced strength and increased risk of fracture caused bydecreased trabecular number and connections associatedwith aging and diseases,(63) such as osteoporosis. In thisstudy, new trabeculae did not form away from the growthplate, even at a young age, providing support for the hy-pothesis that trabeculae are not reformed once lost. Theloss of connectivity is often thought to arise from resorptionof trabeculae. Interestingly there was an early rapid de-crease in ConnD for all strains and sites, including the BALmice where TbN was maintained. In this case, decreasedconnectivity was associated with the transformation of rodsinto plates and not resorption of trabeculae, highlightingthe need for careful interpretation of changes in ConnD.

Mineralization patterns were similar for the three strainsof mice based on relative TMD but not BMD. BMD variedtemporally and in magnitude by site and strain, whereasTMD values provided a much simpler pattern with similarmineral accumulation for all sites and strains. This finding iscomparable to those of Rubin et al.,(14) who found mineralshape and distribution was similar for B6 and C3H micedespite large differences in BMD. Roschger et al.(64) alsofound constant mineral density distributions for normal in-dividuals despite ethnic groups, skeletal site, age, and sex.In our study, the plateau of TMD values after peak struc-tural values is reasonable and within the normal 3- to 6-molag for new bone to achieve complete mineralization.(65) Inaddition, our results overlap with those of a previous re-port,(13) which together provide a complete mineralizationcurve (Eq. 1) and a means of adjusting threshold valueswhen analyzing growing bones by �CT.

Structural adaptations corresponding to strength changesat the tibial diaphysis occurred more quickly in the B6 andC3H mice than BAL mice. In the B6 and C3H mice, com-

pressive and torsional rigidity (CtA and J) increased sig-nificantly during the period of rapid longitudinal growthand became stable or changed gradually. For B6 mice,changes in torsional rigidity (J) are consistent with thosereported by Somerville et al.,(10) and changes in compres-sive rigidity (CtA) similar to trends for cortical thickness byGlatt et al.(11) In BAL mice, geometric changes weregradual, similar to the architectural trends. Overall, the B6and C3H mice had the highest torsional and compressiverigidities, respectively, whereas BAL mice had the lowestproperties in both cases.

In summary, this study provides a detailed characteriza-tion of normal skeletal development patterns for three in-bred strains of mice commonly used in bone research. Thefindings were consistent with previous data from the litera-ture, and repeated-measures analysis provided novel insightinto the patterns of development. Future studies shouldconcentrate on the period up to 12 wk for growth-relatedchanges, 12–32 wk for mature values, and from 32 wk on-ward for age-related deterioration. For future studies withmice in the mature phase, specific timing should be selectedbased on the mouse strain, site, and region of interest (cor-tical versus trabecular). Furthermore, because of the highstability of the vertebra compared with the tibia (and pos-sibly other long bones), this site is well suited for studyingthe effects of different interventions, particularly if in vivoanalysis is not feasible. Finally, of the strains considered, theBAL mice had the most homogeneous and least variablebone structures, with reasonable proportions of trabecularbone at both the tibia and vertebra. Altogether, this studyprovides critical knowledge of normal bone changes andskeletal milestones necessary to optimally plan and inter-pret bone studies examining the effect of age, injury, anddisease.

ACKNOWLEDGMENTS

This work was supported by grants from the AlbertaHeritage Foundation for Medical Research, Natural Sci-ences and Engineering Research Council of Canada(NSERC), the Canadian Institute of Health Research(CIHR), and the Alberta Ingenuity Fund (AIF). The au-thors acknowledge the support of Dr Tak Fung for assis-tance with the statistical analysis and Dr Heather Macdon-ald for insight in bone development.

TABLE 2. ONE-WAY ANOVA OF GEOMETRIC PARAMETERS OF THE TIBIAL DIAPHYSIS BY STRAIN FROM 6 TO 48 WK OF AGE,ASSESSED BY IN VIVO �CT

Outcome C3H/HeN (mean ± SE) BALB/C (mean ± SE) C57BL/6 (mean ± SE) Site F (df) [p]

J (mm4) 0.374 ± 0.001*† 0.317 ± 0.001†‡ 0.437 ± 0.001*‡ 69.6 (2,32) [<0.001]CtA (mm2) 1.106 ± 0.001*† 0.961 ± 0.001†‡ 0.948 ± 0.004*‡ 22.6 (2,32) [0.034]MA (mm2) 0.188 ± 0.001*† 0.309 ± 0.001†‡ 0.447 ± 0.002*‡ 65.8 (2,32) [<0.001]TA (mm2) 1.294 ± 0.001† 1.270 ± 0.001† 1.443 ± 0.002*‡ 37.1 (2,32) [<0.001]

Significantly different (p < 0.02) than *BALB/C, †C57BL/6, and ‡C3H/HeN.J, polar moment of inertia; CtA, cortical area; MA, medullary area; TA, total area.

MURINE SKELETAL GROWTH AND ARCHITECTURAL STABILITY 2057

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Address reprint requests to:Steven K Boyd, PhD

Department of Mechanical and ManufacturingEngineering

University of Calgary2500 University Drive NW

Calgary, AB T2N 1N4, CanadaE-mail: skboyd@ucalgary.ca

Received in original form February 25, 2008; revised form May 15,2008; accepted July 31, 2008.

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