Adaptive Changes in Trabecular Architecture in ... - Science Direct

Bone Vol. 19, No. 1 July 1996: l-8

ORIGINAL ARTICLES

Adaptive Changes in Trabecular Architecture Functional Strain Patterns and Disuse A. A. BIEWENER,’

N. L. FAZZALARI,’

D. D. KONIECZYNSKI,’

in Relation to

and R. V. BAUDINETTE3

’Department of Organismal Biology and Anatomy, The University of Chicago, Chicago, IL, USA 2 Division of Tissue Pathology, Institute of Medical and Veterinary Science, Royal Adelaide Hospital, Adelaide, South Australia 3 Department of Biological Sciences, Flinders University, Adelaide, South Australia

to correlate trabecular architecture with recorded patterns of functional in vivo strain within overlying cortical bone, using the calcaneus of the potoroo (a small marsupial) as a bone model. An attractive feature of the calcaneus in this, and other species in which the calcaneus does not contact the ground, is the simple and highly regular cantilever-like loading that it experiences in transmitting Achilles tendon force to extend the ankle during gait. Because of its fairly simple architecture and mechanical function, previous studies have used the calcaneus to examine functional patterns of cortical bone strain in relation to underlying trabecular architecture,” or the response of cancellous bone tissue to disuse.203’0 However, these earlier studies did not quantitatively relate trabecular alignment with the directions of principal strain or determine changes in trabecular architecture subsequent to disuse. Consequently, in the present study we explicitly test the hypothesis that trabeculae are aligned with the orientation of principal strains produced by functional activity, based on quantitative assessment of trabecular alignment and calculations of principal strains within the overlying cortical bone. Certainly the majority of studies of trabecular alignment and cancellous bone density in relation to functional loading have been carried out on the proximal human femur, using finite element methods to model the distribution of load transmission and its effect on the internal trabecular architecture of the bone. ‘.43’0,‘4,3’,35 Other studies’*,*’ have examined trabecular architecture within the human patella in relation to a modeled distribution of principal stresses associated with load transmission via the quadriceps musculature, in which patellar loading was presumed to be less complex and variable compared with that of the proximal femur. In the former study, determination of mechanical properties within subregions of the patella were well correlated with trabecular architecture and modeled stress trajectories. Other studies have also investigated trabecular architectural changes of the human proximal femur, lumbar vertebrae, and iliac crest in relation to age-related”~2’~24~37 and diseaserelated’,” processes. In these studies, patterns of trabecular bone loss associated with aging or disease appear to arise most generally through reductions in trabecular thickness and the number of trabecular elements. In light of these studies on human bone tissue, we examine the response of cancellous bone within the potoroo calcaneus to a loss of mechanical function by means of Achilles tenotomy, an approach used previously to study the response to disuse of bone mineral content in the sheep calcaneus3” and cancellous bone turnover in the rat.36 While we anticipate that cancellous bone will be lost due to the thinning and loss of trabeculae, we also test

Principal strains and their orientation, determined from in vivo and in situ strains recorded from the lateral cortical surface of the calcaneus of potoroos (a small marsupial) during treadmill exercise and tension applied via the Achilles tendon, were compared with the underlying trabecular architecture and its alignment to test Wolff’s “trajectorial theory” of trabecular alignment. In vivo and in situ principal compressive strains (-800 to -2000 pe) were found to be aligned (mean 161 f 7”) close to the preferred alignment (160”) of underlying trabeculae within the calcaneal metaphysis [a second trabecular arcade was closely aligned (70”) with the direction (71”) of principal tensile strain]. This finding represents quantitative verification of WolFs trajectorial theory of trabecular alignment. These trabecular alignments, as measured by trabecular anisotropy (TbAn, the ratio of horizontal: vertical intercepts), remained unchanged C.p > 0.05) after 8 weeks of disuse. However, trabecular bone volume fraction (BV/TV, -35%), trabecular thickness (TbTh, -25%), and trabecular number (TbN, -16%) were reduced for the tenotomized calcaneii relative to their contralateral controls (p c 0.001 to < 0.003). The reduction in trabecular number was associated with a corresponding increase in trabecular spacing (TbSp, +30%). Together, these results suggest that once trabecular alignment is established during growth (along the directions of principal strain during locomotion), it is not altered when functional strains are removed. (Bone 18:1-B; 1996) Key Words: Principal strain; Wolff’s law; Trabecular bone; Remodeling; Disuse.

Introduction It has long been thought that the distribution of principal strain trajectories engendered through functional activity is responsible for the development and maintenance of trabecular alignment and cancellous bone density within bone.‘6.28,38 Though commonly accepted and popularized as “Wolff’s law,” this trajectorial theory of trabecular organization has been tested by few experimental data. The goal of the present study, therefore, was

Address for correspondence and reprints: Dr. Andrew A. Biewener, Ph.D., Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th St., Chicago, IL 60637. E-mail: aabl @biomorph.uchicago.edu 0 1996 by Elsevier Science Inc. All rights reserved.

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A. A. Biewener et al. Adaptive changes in trabecular architecture

the hypothesis that, following disuse, this loss of trabeculae will result in a loss of trabecular alignment with trajectories of preexisting functional principal strain. Evidence from studies of trabecular alignment in the proximal tibia of osteoarthritic patients” and the proximal tibia of dogs instrumented with a tibia1 knee arthrosis” demonstrate that trabecular realignment occurs in response to altered directions of principal load (strain). Finally, in so far as cancellous bone of potoroos and other marsupials has received little attention in comparison with placental mammal bone, we also evaluate the use of marsupials as a potential animal model for studying bone adaptation to changes in mechanical function. A potential value of marsupial models to study bone growth in relation to changes in physical activity is the extended period of development as a highly immature neonate in an external pouch,‘4 which provides ready access to experimental manipulation at a much earlier developmental stage than the fetus of placental mammals. Materials and Methods Twelve adult male and female potoroos (P. triductylus; body mass range: 0.87 to 1.35 kg) were used in the study. Animals were obtained from breeding colonies housed in a large outdoor enclosures at Flinders University. Animals were divided into two groups: Control animals (four male, two female) were trained over a six day period to run on a motorized treadmill (1.2 x 0.25 m bed). Following training, a rosette strain gauge was surgically attached to the lateral aspect of the left calcaneus of these animals (details of procedure described below) to record functional strains engendered by in vivo locomotor activity. Control animals were sacrificed immediately after completion of all strain recordings, and the calcaneii dissected to confirm gauge placement and orientation. Disuse animals (two male, four female) had an S-10 mm length of the right Achilles tendon surgically excised to isolate the calcaneus from functional load-bearing. Following both surgical procedures, animals were housed as separate groups in 3 x 5 m indoor/outdoor cages. Tenotomized animals were housed for a period of 8 wks before being sacrificed. Because no strain recordings were made of the tenotomized animals, we cannot confirm that their calcaneii were completely unloaded. Video recordings of the tenotomized animals’ gait showed that they used their experimental hindlimb in support, but that the calcaneus remained out of contact with the ground (as it is normally positioned) in most instances during gait (>90% of the strides observed on videotape). In most other aspects (relative time of contact, stride frequency, and speed of walking versus trotting), the animals’ gait appeared normal. To investigate the dynamics of bone turnover and lamellar organization of trabecular bone in this marsupial species, all animals were given double fluorochrome labels administered on two successive days, using a lo-day interval between labels (First: Ledermycin, 15 mg/kg ip; second: calcein, 15 mg/kg ip) prior to sacrifice. These labels were then analyzed under flourescence microscopy (see below). Surgical Procedures To attach the rectangular rosette foil strain gauge (4-mm-diam gauge backing, type FRA- 11, Tokyo Sokki Kenyujo) to the lateral surface of the calcaneus, control animals were anaesthetized via a mask with isofluorane. After shaving and cleansing (Betadine scrub and solution) the lateral aspect of the distal limb, a 1.5 cm incision was made from the base of the calcaneum superiorly along a line just anterior to the Achilles tendon. The overlying soft tissues were reflected and the underlying mineralized surface

of the calcaneus exposed by removal of the periosteum using a scalpel. Hemostasis was achieved by electrocautery. The cortical bone surface was lightly scraped using a periosteal elevator and dried with ether, applied via a cotton-tipped applicator. The strain gauge, which had been previously sterilized in a UV cabinet, was then bonded to the lateral calcaneal surface using methyl-cyanoacrylate adhesive applied to its undersurface by pressing down on the gauge with a greased cotton-tipped applicator. The lead wires were then passed subcutaneously to an opening in the skin located anterior to the hip and soldered to connector pins that were mounted into a sterile plastic connector (Amphenol, type 222). The pins were insulated within the connector using silicon rubber adhesive (RTV, Dow Coming) and the connector sutured (0 silk) to the skin. The base of the connector was sealed with additional application of RTV adhesive at the skin’s surface. The opening at the strain gauge attachment site was then closed using 3-O silk suture. Tenotomized (disuse) animals were prepped for surgery, anesthetized, and their wounds sutured following the same procedures as described for group A animals. Both groups of animals were administered an analgesic (3 mg Flunixin, Schering-Plough) and an antibiotic (20,000 IU procaine penicillin) following surgery. Animals were allowed to recover full consciousness before being returned to their cages.

In Vivo Bone Strain Recording Procedure Following a 6 to 24 h period of recovery from surgery, group A animals were run on a treadmill over a range of speeds (0.4 to 1.5 m/s) that included walking and slow galloping gaits. Strains from the rosette strain gauge were conditioned and amplified via shielded lead wires connected to a Vishay Measurements Group model 2100 bridge amplifier. The strain signals from the amplifier were sampled on a 486computer via a Metrabyte DASH16F 12-bit analog/digital converter using custom designed data acquisition ASYST software at a resolution of 3.25 &bit. Principal strains and their angle to the longitudinal axis of the calcaneus (Figure 1) were then calculated from the digitized raw strain signals, based on standard formulae that assume a planar state of strain.’ Following experimental recordings, the animals were sacrificed (500 mg pentobarbitone sodium injected intravenously) and examined to verify secure attachment of the strain gauge to the bone’s surface. In three of the six animals this was the case. However, in the other three animals the gauges were either unattached or not securely bonded, consistent with the low magnitude and irregular patterns of strain recorded in these animals. As an alternative to making in vivo strain recordings in these animals, we conducted in situ strain recordings to simulate loading of the calcaneus by means of tension applied via the Achilles tendon.

In Situ Strain Recording Procedure Following attachment of a new rosette strain gauge to the lateral surface of the calcaneus of these animals, in situ rosette strain recordings were carried out by tying 0 silk to the proximal Achilles tendon and connecting this to an isometric force transducer. With the foot positioned against the countertop and the metatarsophalangeal and ankle joints stabilized at flexion angles (135” and 90”, respectively) characteristic of their position during limb support when peak in vivo strains were recorded, tensile forces (up to 40 N) were then applied to the Achilles tendon via the force transducer. In situ strains were sampled at 200 Hz for a series of six cycles. This procedure was repeated with the ankle

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Talccalcaneal Joint Reaction Force & Moment

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A. A. Biewener et al. changes in trabecular architecture

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I

-

--

-

L. Axis

X -axis

b

Y -axis

Figure 1. (a) Radiograph of potoroo hind foot showing rosette strain gauge position on the lateral aspect of the calcaneus in relation to underlying trabeculae. The small solder disk and lead wires from the gauge were used to establish the gauge’s orientation relative to the longitudinal axis of the bone. (b) Schematic drawing of the left calcaneus (lateral view) of a potoroo showing the definition of principal compressive (E,) and tensile (E,) strain angles (bold arrows) used for comparison with trabecular alignment, as well as the external forces that are applied to the calcaneus by the Achilles tendon and by joint reaction forces at the proximal end of the bone. The horizontal or longitudinal structural axis of the bone (L. Axis) was defined by the superior-most surfaces of the bone, at its proximal articulation with the talus (also referred to as the astragalus) and at its distal end where the Achilles tendon inserts

positioned

at 70” and 110” to test for variation in principal

orientation

with change in ankle flexion angle.

Bone Sectioning, Flourescence Stereology Procedures

Microscopy,

strain

and

Following in vivo or in situ strain recordings, the calcaneii were cleaned of soft tissues and radiographed (Hewlett-Packard Faxitron) with the rosette strain gauges intact to determine the alignment of the gauge axis relative to the bone’s longitudinal axis (Figure la). A small (0.8 mm) solder “disk,” which had been previously glued to the gauge’s top surface aligned with the E* gauge axis, was used to measure the angle of the rosette strain gauge relative to the longitudinal axis of the calcaneus (parallel to the superior border of the distal calcaneus at the Achilles tendon insertion site and the proximal calcaneus at the tibiocalcaneal joint, Figure 1b). The bone specimens were then fixed in 10% formalin, vacuum embedded in methacrylate and sectioned to 10 pm thickness. Two midsagittal longitudinal sections were obtained from each bone specimen for flourescence microscopy and stereological analysis. Assessment of BMU-based bone

turnover characteristics and the histological organization of trabeculae were analyzed using a flourescence microscope at 82.5x magnification. Trabecular bone volume fraction (BV/TV, %), trabecular thickness (TbTh, mm), trabecular spacing (TbSp, mm), and trabecular number (TbN) (terminology following 25) were measured for each of the tissue sections using a Quantimet 520 image analyzing computer system (Leica Cambridge Ltd., England).26 In addition, an index for trabecular anisotropy (TbAn) was calculated as the ratio of horizontal to vertical intercepts of trabeculae. This index differs from that used by Kuhn et al.” and Goulet et al.,” derived from measurements of mean intercept length of test lines projected at differing orientations onto the trabecular structure. Our index for TbAn is comparable to the resulting “best-fit” line describing trabecular orientation by this latter method. TbAn was determined with the alignment of the calcaneus referenced to the compressive principal strain axis (161’) or equivalently -19”) measured from in vivo and in situ strain recordings; hence, increasing TbAn (i.e., increasing horizontal intercepts relative to vertical intercepts) indicates increasing alignment of trabeculae with the compressive strain axis.

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architecture

In vivo principal strains 0 1 .l m/s

180 Q s ‘E P

120 60 -

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Time (set)

B.

In situ pulls via Achilles tendon

-2000 ’

0 Figure 2. Representative (a) polarized light and (b and c) fluorescence micrographs obtained from longitudinal sections of cancellous bone in the potoroo calcaneus, showing (a) the lamellar organization of trabeculae within the calcaneus and (b and c) the incorporation of fluorescent label into new bone over a I O-day interval (Ll and L2). Lamellar deposition and focal osteoclastic resorption (arrows in b and c) of preexisting label parallels the BMU based bone turnover observed generally within placental mammals. Scale bars: 100 km.

1

2

3

4

5

Time (set) Figure 3. Representative

principal strains and the angle (Phi) of principal compressive strain that were calculated from (a) in viva and(b) in situ recordings made from rosette strain gauges attached to the lateral aspect of the calcaneus. Six cycles of in viva limb support are shown in (a), as the animal walked on the treadmill at 1. I m/s. Three strain loading cycles produced by tension applied via the Achilles tendon in situ are shown in (b). Horizontal bars depict the stance phases during gait or the in situ

loading phases. TbAn with the calcaneal bone specimen oriented at angles ranging from 0” to 180” to test for maxima and minima in TbAn. Because we wished to correlate the “global” alignment of trabeculae within the calcaneus to the surface measured principal strains, as well as to compare global changes in trabecular architecture of control versus disuse calcaneii, a circular sampling frame encompassing the maximal area

This

was assessed

by measuring

of trabecular bone within each section, excluding bordering regions of cortical bone, was used to avoid bias of regional differences in location and orientation among specimens. Paired comparisons were made between the tenotomized and control contralateral hindlimb to assess the effect of disuse on trabecular bone morphology. Results are presented as the mean f SD.

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0.6

0.4 Principal & Angle 0 60

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120

140

160

160

Angle (degrees)

Results BMU Based Remodeling

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anisotropy (TbAn) measured as the ratio of horizontal line intercepts to vertical line intercepts (Id I,,) plotted as a function of specimen orientation (N = 6). A value of one (horizontal line) indicates no preferred orientation of trabecular elements (i.e., isotropy). Maxima in TbAn are consistently observed at a specimen angle of approximately 160”, corresponding to the mean orientation of compressive principal strain measured in viva during gait and in situ (see Figure 3). Similarly, minima in TbAn are observed near 70”, matching the orientation of tensile principal strains that were measured. Angles of 0” and 180” correspond to the horizontal or longitudinal anatomical axis of the bone (see Figure 1).

c

= c

changes

Figure 4. Trabecular

Tensile Principal E Angle

1.6

2 .

Adaptive

Trabecular Bone

Figure 2 shows representative polarized light (Figure 2a) and flourescence (Figures 2b, c) micrographs of trabecular bone in the potoroo calcaneus, illustrating the lamellar organization of trabeculae, similar to that of placental mammal bone. Labels 1 and 2 correspond to a IO-day interval just prior to sacrifice. Incorporation of new label by means of lamellar deposition on trabecular surfaces and osteoclastic resorptive activity of preexisting label (arrows) are indicative of focal BMU-based tumover kinetics similar to that observed for mammalian bone generally.’ An average mineral apposition rate (MAR) of 1.2 f 0.2 pm/day and a formation period (or) of 25 f 4 days were measured in control calcaneii. Principal Strain Angle in Relation to Trabecular Orientation In vivo (Figure 3a) and in situ (Figure 3b) compressive principal strains measured at the lateral surface of the calcaneus in the range of -600 to -1200 p,e were consistently oriented at a mean angle of 161 + 7” to the longitudinal axis of the calcaneus in all six animals (range: 150” to 169”). The mean orientation of com-

A.

pressive principal strain during in vivo treadmill activity (165 & 6”, measured at peak strain) was not significantly different (p > 0.05) from that during in situ loading via the Achilles tendon (158 * 9”). For both in vivo and in situ loading, the orientation of principal strain was consistently maintained throughout most of the loading cycle (stance phase of gait, or tension applied via the Achilles tendon); however, shifts in principal strain angle are common during the unloaded, or swing phase of gait. Trabecular anisotropy (TbAn) of cancellous bone deep to the lateral cortical surface was maximal at an orientation of 160”, consistent with the orientation of compressive principal strain (Figure 4). The minimum in TbAn corresponds to the orientation of trabeculae that were aligned with the principal tensile strain axis (70”). Trabecular Architecture

Changes with Disuse

When comparing tenotomized calcaneii with their contralateral controls, 8 wks of disuse resulted in significant changes in all architectural parameters, except trabecular anisotropy (Figure 5 and Table 1). Trabecular bone volume fraction (BViTV) decreased overall by 35%, largely attributable to a 25% decrease in trabecular thickness and a 16% decrease in trabecular number. These decreases resulted in a 30% increase in trabecular spacing (Table 1 and Figure 6). In contrast, despite the loss of elements

B. I-

0.6

0.15

* Contralateral control

0.4

0.10

2

1

0.2

TbAn

BVKV

TbTh (mm)

TbN (#/mm)

Figure 5. Histograms of (a) trabecular anisotropy (TbAn) and bone volume fraction (BVmV), and (b) trabecular thickness (TbTh) and trabecular number (TbN) in tenotomized (disuse) and contralateral control calcaneii. Error bars indicate *l SD. Asterisks denote significant differences for paired comparisons at p < 0.003. Values are reported in Table 1.

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Table 1. Stereological Statistical = 12) Group Tenotomy Contralateral control

measurements (mean ? SD) of tenotomized and contralateral control calcaneii. tests based on Student’s t-test for paired comparisons (of two sections per specimen, N

BVrJV

TbTh (mm)

TbSp (mm)

0.130 (kO.030) 0.200 (kO.027) p < 0.0001

0.073 (kO.014) 0.097 (kO.019) p < 0.0001

0.502 (kO.077) 0.387 (+0.050) p < 0.000

in tenotomized calcaneii, no significant change in trabecular alignment (TbAn) was observed (Figure 5a and Table 1). Discussion The results presented here support previous observations that the underlying trabeculae within the sheep calcaneus are oriented with the principal strain direction in the cortical surface.” Using stereological techniques, we quantitatively show that trabeculae align most closely with the compressive (and tensile) principal strain axis recorded in the overlying cortical bone. In the earlier study, this was determined by examining radiographs by eye. Hence, assuming that the orientation of principal strains in the cortical surface reflects the orientation of principal strains within the underlying cancellous bone, our finding provides quantitative verification of Wolff’s”8 trajectorial theory of trabecular orientation. Because the calcaneus is subjected to relatively simple cantilever-like loading by the Achilles tendon during gait, it represents an ideal element for testing the correlation between func-

A. Tenotomy

5mm B. Control

Figure 6. Radiograph prints of a tenotomized calcaneus (a) and its contralateral control (b), illustrating the loss and thinning of trabeculae that results following an 8 week period of disuse. No significant realignment, or disordering, of remaining trabeculae is observed compared with the normal principal trabecular alignment that develops during growth (see Figures 4 and 5).

I

TbN (#/mm)

TbAn (1,/I,)

1.763 (+0.216) 2.100 (kO.269) p < 0.003

1.845 (kO.157) 1.883 (?0.098) p > 0.05

tional principal strain orientations and trabecular alignment. It seems likely that the uniform orientation of the Achilles tendon with respect to the calcaneus promotes a highly regular loading environment and results in highly regular directions of principal strain. Thus, for this bone element the principal trabecular alignment may be expected to be orthogonal as well. In contrast, bone elements that may be expected to experience a wider range of load distribution and loading orientation at their articular surfaces would likely show more variable principal strain trajectories and hence, would be less well correlated with trabecular alignment. This is illustrated by results of a finite element model of the human femur, in which Carter et aL4 found that trabecular alignments predicted by multiple-load cases were not necessarily orthogonal and did not correspond to the directions of principal stress for any single load case. Our finding that eight weeks of disuse results in an overall reduction in trabecular bone volume fraction is consistent with disuse resulting in a generalized loss of bone mineral content within the calcaneus of sheep” and in the femoral metaphysis of rats.‘6 In the present study, the observed 35% decrease in BViTV was mediated by significant decreases in trabecular thickness (25%) and number (16%). A similar pattern of bone loss has also been observed recently in the proximal tibia of rabbits, following a proximo-medial osteotomy designed as an experimental model of osteoarthritis.32b However, in both the potoroo calcaneus and the proximal tibia of rabbits the fundamental structural alignment of the remaining trabeculae was not altered, counter to our hypothesis that a loss of functional loading would result in a loss of trabecular alignment. Results consistent with our findings and interpretation have also been reported for the distal metacarpal of immobilized forelimbs in dogs; 12a in which the trabecular thicknesses of both vertical and horizontal trabeculae experienced similar rates of change, suggesting no loss of trabecular ahgnment. In a recent study of disuse in the calcaneus of sheep, following external immobilization of the ankle,‘” daily bouts of walking exercise that briefly interrupted much longer periods of disuse was insufficient to prevent significant bone loss (21%). Consequently, while functional exercise may be important for maintaining bone mass and cancellous architecture, a certain intensity and/or duration of daily exercise must occur to have an effect. Based on the similarity of the pattern and magnitude of bone loss in the cancellous bone of this marsupial species with that observed in other placental mammal species subjected to disuse, including man,9.‘3,22 our observations of the lamellar nature of bone formation and BMU-based remodeling within the potoroo calcaneus support the use of this, and possibly other marsupials, as a model system for investigating early skeletal development in response to exercise and mechanical loading. The similarity of principal compressive strains that we recorded during functional locomotor activity (-600 to -1200 FE) with those recorded in the calcaneus of sheep’s and the long bones of other placental mam-

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mals 3,19329and birds* during moderate treadmill exercise also lends support for the use of marsupial species in bone remodeling studies. The mineral apposition rates and formation sigma that we observed for the potoroo calcaneus, in particular, match well the values obtained for rodents.36 The 25 day formation period (ur) for the potoroo calcaneus, moreover, indicates that the absence of any loss in trabecular alignment was not due to an insufficiency in the period of disuse (8 weeks) employed in the present study. Instead, it seems possible that trabeculae may be formed and align in response to principal strain magnitudes and directions experienced during growth and that the resulting cancellous structural alignment, once established, constrains subsequent realignment of the trabeculae to changes in functional loading imposed at maturity. Thinning and loss of trabecular elements following an extended period of disuse would, therefore, not be expected to change the alignment of the pre-existing structure. This interpretation is consistent with prevailing views on th& 4 mechanism of trabecular bone loss in age-related osteopenias, in which apposition and resorption of bone mineral on surfaces of aligned trabeculae leads to perforation and ultimate loss of trabecular elements but does not alter trabecular alignment. Singh et a1.33 do not describe, as a characteristic feature of osteoporosis, changes in trabecular alignment for advanced osteoporotic patients; instead, trabecular alignment appears to persist in the remnants of cancellous bone structure. Whether this holds for exercise-induced changes in principal strain patterns in mature animals, potentially stimulating trabecular realignment, remains to be demonstrated. Studies of trabecular orientation, following alteration of canine metaphysial loading by means of an instrumented porous-coated tibia1 knee ar throsis2i and in association with osteoarthritis of the human knee,15 indicate that trabecular realignment is possible and does occur. Finally, if during growth trabecular alignment is regulated by the directions of principal strains engendered by functional loading, then removal of functional loading through disuse during growth would be expected to result, not only in trabecular thinning and a loss of trabecular number, but a loss of trabecular alignment as well. We plan to test this hypothesis in future studies of marsupial pouch young.

Acknowledgments: The authors thank Peter McNiel for processing

bone specimens, Dale Caville for photography, Ian Parkinson for help with stereological techniques, and Dr. Mitch Schaffler for comments on an earlier draft of the manuscript. This work was supported by grants to A.A.B. (Grant No. NIH-AR39828) and R.V.B. (Australian Research Council).

References 1. Beaupre, G. S., Orr, T. E., and Carter, D. R. An approach for time-dependent bone modeling and remodeling application: a preliminary remodeling simulation. J Orthop Res 8:662+570; 1990. 2. Biewener, A. A., Swatz, S. M., and Bertram, J. E. A. Bone modeling during growth: dynamic strain equilibrium in the chick tibiotarsus. Calcif Tiss Int 39:390-395; 1986. 3. Biewener, A. A. and Taylor, C. R. Bone strain: A determinant of speed and gait? J Exp Biol 123:383400; 1986. 4. Carter, D. R., Orr, T. E., and Fyhrie, D. P. Relationships between loading history and femoral cancellous bone architecture. J Biomech 22:231-244; 1989. 5. Dally, J. W. and Riley, W. F. Experimental Stress Analysis. New York: McGraw-Hill; 1978. 6. Fazzalari, N. L., Darracott, J., and Vernon-Roberts, B. A quantitative description of the cancellous bone in the head of the femur using automatic image analysis. Metab Bone Dis Relat Res 5:119-125; 1983.

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Date Received: December 19, 1995 Date Revised: March 8, 1996 Date Accepted: April 8, 1996