BoneandMineruI, ll(l990) 111-122 Elsevier

BAM 00309

111

ontuori,

Centro de Estudios de Metabolismo Fosfocdlcico (CEMFOC), Facultad de Ciencias MPdicas, Universidad National de Rosario, Santa Fe 3100,200O Rosario, Argentina

(Received 14 December 1989) (Accepted 19 June 1990)

The effects of i.p. doses of 0.016,0.16,1.6,5, 16,50 and 160 ,uWkglday of APD, over a period of 23 days, on geometric and biomechanical properties of femoral diaphyses in bending were determined in groups of seven growing rats.

Both elastic and ultimate strength increased with low doses and decreased with high doses. Geometric (mass) variables (diaphyseal volume, walYlumen ratio) correlated positively, and material properties (limit elastic stress, modulus of elasticity) negatively, with log dose. Normal mass and improved quality at low doses, and improved mass and impaired quality data at high doses were obtained. No changes in sectional moment of inertia (Ix, an expression of bone architecture) were observed.

Biphasic changes in diaphyseal strength expressed the effects of APD on material quality in spite of mass variation. The contrasting lack of changes in Ix may have reflected the blocking effect of APD on osteoclast-osteoblast communication, essential for directional modulation of remodelling.

_ __-___________.-

Key words: Amino-hydroxy-propylidene-bisphcsphonate; Femur; Rat; Bone biomechanics; Cortical bone

Bisphosphonates are known to (a) inhibit precipitation of calcium phosphate, slow- ing down the aggregation and dissolution of crystals because of their marked affini- ty to the solid phase, (b) inhibit bone calcification through undetermined mecha-

Correspondence to: Prof. Jose L. Ferretti, MD, PhD, JuanB. Just0 1427,200G Rosario (SF), Argentina.

0169~6009/90/$@3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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nisms and (c) inhibit bone resorption and the coupled bone formation by affecting cellular mechanisms concerning osteoclast recruitment, macrophage activity and cytokine production [l-3]. A dependence of these actions on dose-time schedule and kind of compound assayed [4-71 obscured the interpretation of the clinical rele- vance of bisphosphonate therapy when used for controlling bone metabolic disor- ders such as Paget’s disease and osteopenic states.

Notwithstanding the usefulness of bisphosphonates for treatment of skeletal dis- eases, reports dealing with a functional, biomechanical description of their effects on bone are scarce and inconclusive. Chan et al. [8] reported in 1977 that daily S.C. administration of 10 (but not 5) mglkglday of APD during 20 days reduced diaphy- seal diameter, wall thickness, and torsional ultimate strength of chick femurs. More recently, Glatt et al. [9] found that S.C. injections of 0.1-1.0 mg/kg of APD per week from the 13th to the 65th week of age improved femoral ultimate strength in mice. Preliminary data from one of us (EM, unpublished) showed that there was also a 52% increase of compressive strength of rat vertebrae after oral treatment with 8 mglkglday of APD during 30 days. These results are difficult to correlate with each other unless precise data on material properties are also known and a more comprehensive dose-range is assayed in longer studies.

The present report describes the effects of the 25-day administration of the whole range of useful doses of APD (regarded as the parent compound among the bis- phosphonates currently in use [3]) on geometry and mechanical structural (whole diaphyses) and material (bone tissue) properties of growing rat femurs as deter- mined by bending tests at low strain rates [lo-131 suitable for comparison purposes [ 14,251. Results are discussed in relation to a most comprehensive study on APD ef- fects on rat long bone histology, biochemistry and metabolism carried out within a similar period by Reitsma et al. [ 161 whose dose-range and schedule of administra tion were reproduced in order to allow for a reliable reference. Evidence is shown for biphasic effects on diaphyseal mechanical performance. Discussion affords not only an explanation for the above referred controversy, but also a rationale to inter- pret the skeletal repercussion of APD administration at low and high doses from a mechanical point of view.

Eight groups of seven male IIM [17] rats of 35-40 days of age and weighing about 80-100 g at the start of the experiment were housed in metabolic cages under natu- ral light-dark photoperiod in a temperature-controlled (23 “C) room. Animals were fed a semisynthetic diet adequate for growing rats (87% total wheat flour, 10% ca- sein, 3% salts mixture [18], Ca/P content = 1.0/0.8%) ad lib. and given daily i.p. in- jections of 0.0045,0.045,0.45,1.4,4.5,14 or 45 mg/kg (0.016-160pMlkg) of APD dissolved in saline, or solvent alone (controls) during 25 days.

At the end of experiment animals were sacrificed by ether overdose and both dis- sected femurs were immediately submitted to 3-point bending tests as described be- fore [ll-133. The bones, with their anterior aspect facing down, were placed laying

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freely on two supports separated by a constant distance L = 83 mm and equidistant from their ends. As the whole bone length range from 28.5 to 31.7 mm, the chosen value for L (scarcely more than 215 of the mean bone length) avoided any influence of changes in metaphj:eal architecture and minimized the experimental error de- rived from the small deviations from regularity usually shown by the diaphyseal sec- tion throughout the central part of the shafts. Each bone was centrally loaded at a rate of 10 N/mm in order to obtain the load (W)/deformation (d) curves showing both the elastic (linear) and plastic resistance components until fracture, separated by the yielding point (departure from linearity) [15]. As bone segments between supports (hollow, elliptically shaped cylinders) were reasonably straight and uni- form in section throughout, the micromorphometrical determination of the hori- zontal and vertical, external (k&B) and internal (h,b) diameters of the fracture sec- tions enabled calculation of three diaphyseal geometric properties:

Volume of bone between the supports = L n ( amount of tested diaphyseal bone mass.

- MI), an indicator of t

Wall/lumen ratio of the mid sections of bone shafts = l/2 [N - h)/h + (B - b)lb], an indicator of the relative intensity of periosteal apposition and endosteal re- sorption.

Second moment of inertia (Ix) of the fracture sections in relation to the hori- zontal axis = x ( - b3h)/64, an estimator of the efficiency of spatial distribu- tion of bone material throughout the section (bone architecture) related to the sense of action of the most common challenging stresses in this case.

Graphic analysis [lo] of the WM curves determined the following.

a. Structural (whole bone) mechanical properties: maximum elastic strength (W,,, load at the yielding point); ultimate strength (load at fracture); maximum elastic deformatiion (d,,, arrow of the arch formed by the bending bone at the yielding point); stiffness (W&f,,, load/deformation ratio, slope of the W/d curve during elastic behavior).

b. Material (bone tissue) mechanical properties (intrinsic quality indicators which allow comparison of bones of different size andlorshape):

maximum elastic stress or strength = L. Be We,/8 Ix; @ Young’s modulus of elasticity (stress/strain ratio, estimation of tissue stiffness) =

W,, m L3148 d,,-lx .

Values of geometric and structural variables, previously shown to linearly correlate with body weight (b.w.) [12], were considered both in crude form and statistically adjusted to 150 g (the b.w.-point of convergence for each variable as previously de-

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termined in rats from different strains) in order to assess and minimize the influence of any effect of treatment on growth. Data were averaged for each animal and group. Standard statistical analyses were carried out for evaluation of the data [ 191.

Results

Mechanical performance of the whole diaphyses Table 1 shows raw values for final b.w. and fracture load, load at the yielding point, and stiffness of the disphyses. Only the highest dose affected significantly the bio- mass of the animals with respect to controls, so that statistical adjustment of whole data of the three structural variables considered to a common, 150 g b.w. (Fig. 1) did not alter the general sense of the differences induced by treatment.

Biphasic effects on limit-elastic and ultimate strength were observed. As as- sessed from either raw data or b.w.-adjusted curves (Table 1 and Fig. l), these in- creased at low APD doses (0.0045-0.45 mg/kg/day) and were progressively re- duced with log-dose dependence at higher doses, so that values significantly lesser than those of controls were reached at 50 and 160 mglkglday. Stiffness (loadldefor- mation ratio) was not affected by 0.0045 and 0.045 mg/kg/day but was significantly reduced from 0.45 mg/kg/day onwards, especially at the highest dose.

A particular relationship between the two components of the load/deformation ratio, that is, the limit-elastic load (IV,,) and the corresponding deformation regis- tered (d,,), was observed for each treatment. Fig. 2 shows the representative distri- bution areas for the plotted pairs of values of these variables in control and treated groups.

Table 1

Means and SD of raw values for b.w. and three of the bone structural properties (fracture load, load at the yielding point, and diaphyseal stiffness) of femurs from every studied group

APD dose b.w. (g) Fracture load Load at the yield@ Diaphyseal stiffness (mgW (N) point(N) (N/mm) day)

li: SD ii SD t SD ir SD -

0 193.2 23.5 90.8 4.9 80.8 6.8 0.0045 199.2 43.0 iO2.5 ll.ga 93.5 9.ga 0.045 188.3 15.0 95.3 2.7” 88.4 4.0 0.45 193.2 23.2 95.5 14.3 87.4 4.2a 1.4 173.6 25.4 83.6 9.2 75.1 6.3 4.5 170.7 15.9 82.6 4.gb 70.3 3.gb

14.0 170.7 25.6 72.9 s.3c 67.4 6.2b 45.0 118.6 17.4c 49.1 5.9c 43.2 s.gc

121.0 14.7 123.8 12.5 116.5 14.8 97.6 lO.lb 88.4 4.T 92.3 20.Sb 86.1 13.2’ 45.6 9.7’

a, b, and c indicate respectively 0.05, 0.01, and 0.001 significance levels of the difference with respect to controls.

FRACTURE LOAD r N 1

60

60

60

40

100

50

LOAD AT THE YIELDING POINT ( Wet, N )

OIAPWYSEAL SIIPFNESS (Wel/dpl ,N.mm -‘I

APO ‘WWd o 0 0045 0.045 045 14 4.5 I4 45 pM/Kqld 0 0.016 0.16 I6 5 16 50 160

Fig. 1. Means ? SD of fracture load, limit elastically resisted load (We,) and stiffness (W&i,, ratio) of fe- mur diaphyses (adjusted to 150 g b.w.) from every group studied in relation to APD doses. Asterisks (*, *+, ***) respectively indicate 0.05, 0.01 and 0.001 significance levers of the differences with respect to

control group.

40 60 80 100 N

Limit elastic strenqth (Loadat the qietdinq pOint.W,, 1

Fig. 2. Representative distribution zones for load (W,,) and deformation (d,,) pairs of values at the yield- ing point (limit of elastic behavior) of femur diaphyses from control animals (pointed area) and for

groups receiving 0.0045-0.045 (l-2), 0.45 (3), 1. .4-4.5 (4-S). 14 (6), and 45 mglkglday (7) of APD.

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Geometrical properties (values adjusted to a common 150 g b. w.) Both diaphyseal volume and wall/lumen ratio showed a positive, linear correlation with log-dose throughout the range assayed (r = 0.56 and 0.60, P < 0.001, Fig. 3a,b), with predominance of values over the normal f + 1 SD limit from 1.4 to 4.5 mg/kg/day onwards. Differential effects of treatment on internal diameters b and h in relation to external diameters B and H (Fig. 4) maintained constancy of moment of inertia of fracture sections (taken with reference to the horizontal axis) in every oroup (Fig. 5) in spite of the referred positive changes in the other geometric vari- i> ables.

Material mechanical properties Both limit-elastic stress and modules of elasticity of diaphyseal tissue showed a neg- ative, linear correlation with log-dose throughout the assayed range (r = -0.79 and -0.68, P < 0.001, Fig. 3c,d), so that there was a significant tendency to obtain

OIAPHYSEALVOLUME c,iiiJ

a WALL/LUMEN RI10

C LIMIT ELASIIC STRESS tMN.m-’ I

D MODULUS OF ELASTICITY tG.N.m-'1

I 1 I I I I I

APO mq/kq/d .0045 sl45 .45 I.4 4.5 14 45 ,Mkqld ,016 .I6 1.6 5 I6 50 160

Fig. 3. Means f SD of diaphyseal volume (a) and wall/hrmen ratio (b) (adjusted to 150 g b.w.); and bone material limit elastic stress (c) and modulus of elasticity (d) of every group studied in relation with APD doses. Horizontal bands represent ir f 1 SD of control values. Asterisks (*, **, ***) indicate respectively the 0.05,O.Ol and 0.001 significance levels of differences with respect to controls. For coefficients of car-

relation see the text.

-H---

L 4

- h, - v h, ------+

k H b h WLH IX

~rnrn, tmm, tmnl, ,mm, tInal 4,

c 1.ra 3.42 1.62 2.22 .619 3.03

,rf-,)-4LJ 1. Em, (3.421 I.?3 1.71 -795 3.06

A _- -- +6. B% -ZJ. ox +20.4x -0.01%

Fig. 4. Representative sketch shoving crude mean B, Zf (thick line), 6 and h values (bc, kc, thin, contin- uous tine) of sectional fracture area of control animals. Taking profit of the similarity between H/B ratios showed by control and 45mg/kg/day-treated animals (1.24 vs 1.27), the outer ellipse can be also con- sidered as proportionally assimilated to the sectional fracture area of this group. The central ellipse (thin, discontinued line) represents the mean internal measures (b45, h45) of fracture section for these animals, expressed as proportionally related to the external ellipse for comparative purposes in order to avoid any influence of reduced body size in treated rats. Absolute, mean values of B, H, b and h for con- trol (C) and 45mg-treated (APD-45) rats and the resulting walVlumen ratio (WLR) and sectional mo- ment of inertia (Ix) data are given at the bottom, in order to show how the proportional differences (4) registered in b and h significantly affected WLR without any influence on Ix if body size was taken into

account.

SECTIONAL MOMENT OF INERTIA t mm 4 )

l-

W”W’ 0 0.0045 0 045 0.45 I.4 4.5 14 45 APO

,uM/kq/d 0 0016 016 16 5 16 50 160

Fig. 5. Means + SD of sectional moment of inertia of femur diaphyses (adjusted to 150 g b.w.) from con- trol and treated groups in relation with APD doses.

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values higher than normal for the lowest, and lower than normal for the highest doses.

Discussion

The APD effects on diaphyseal stiffness were negative and only evident at high doses (Fig, 1). The biphasic behavior of dose-response curves of APD effects on diaphyseal elastic and ultimate strength (Fig. 1) showed, however, that the sign of final results on this aspect of mechanical performance at the organ level depended upon the intensity of treatment. Understanding this fact should help to explain the apparect contradiction between the few, partial reports available on APD effects on bone biomechanics. The increased ultimate strength obtained at low doses was consistent with Glatt’s findings studying the mechanical properties of whole femora after a moderate, long-term (l-year) treatment with 0.1-1.0 mglkg (0.35-3.5 PM/kg) of APD per week in mice [9]. On the other hand, the impairment of this property observed at high doses is congruent with the reduction of torsional strength shown by Ghan after administration of 10 (but not 5) mg/kg/day of sodium etidronate (EHDP) during 20 days to chicks [g].

These results could be interpreted, in agreement with Glatt [9], as an evidence supporting the usefulness of relatively long-term, moderate-dose APD treatment to enhance long bone mechanical strength. The distribution zones shown in Fig. 2, however, offer a rationale for the limitation of this criteria. In fact, from the rela- tionship between the maximum load elasticity supported by bones and the asso- ciated deformation they underwent (directly derived from load/deformation curves) it may be predicted that only the lowest assayed doses (0.0045-0.045 mg/kg/day) will enhance elastic strength without increasing diaphyseal deformabili- ty. With 0.45 mglkglday similar improvements could be achieved in strength but at the risk of also increasing deformability. Bone deformability is not always asso- ciated with bone fragility or weakness because it is also in direct relationship with the energy elastically absorbed by the deforming bone, which in turn determines the probability of occurrence of cornminute fractures [20]. At 1.4-4.5 mg/kg/day no significant strength changes should be obtained, yet there should be a high risk of reducing stiffness. With 14 mg/kg/day not only stiffness but also strength may be re- duced, and with 45 mg/kg/day both properties will always be impaired in the condi- tions determined by the assayed rat model.

Bone mass indicators (diaphyseal volume, wall/lumen ratio) were positively re- lated to log-dose (Fig. 3a,b), in concordance with the well-known, dose-dependent inhibitory effect of APD on bone resorption in vitro and in vivo [ 1,16,21-261, that was evident with as little as 0.045 mg/kg/day even in short, 6-day studies and was maximal (perhaps complete) at 4.5 mg/kg/day in rats [22]. This effect could also be associated with a reduction of bone formation, considered a secondary phenome- non, delayed from 6 days to 3 weeks in rats, linked to a reduced osteoclast activity through cell-to-cell interactions [22,27]. At any rate, whatever had been the degree of eventual depression of bone formation in our animals (not assessed in this study),

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it seemed not to have been sufficient to neutralize the dose-relate ment resulting from inhibition of bone resorption, as judged fro olume and wall/ lumen ratio changes at high doses. Lack of variation in section ment of inertia (Figs. 4 and 5) in spite of sharp increments in volume and wall/lumen ratio, unlike that observed in many previous studies on rat and chick bone biomechamcs in dif- ferent conditions [ll-13,28-301, should be regarded, however, as a sign of inabili- ty of bone cells to arrange newEy formed bone following an architecturally efficient pattern. This argument could represent the biomechanical counterpart to the col- lected evidence favouring the hypothetical APD blocking effect on resorption-for- mation coupling [3,22,27,31], which is supposed to be essential for an adequate di- rectional modulation of remodelling by the mechanical stimulation of bone.

The negative correlations shown in Fig. 3c,d indicated that bone material proper- ties improved at low APD doses and impaired at high doses with respect to controls. These properties of cortical bone are much affected by its calcium content [l&14, 32-401. Their enhancement at low doses was congruent with the increm Ca/hypro index found by Reitsma [16] in 23-day studies with A

kg/day (0.0045-4.5 mg/k nd in Ca content per unit dry d by Glatt with 0.1-1.0 er week during 1 year in co-

incidence with enhancement of bone strength in mice [9]. Impairment at high doses, on the other hand, corresponded well with a reduction of mineralization reported by Reitsma [ 161 from 40 @Ukg/day (142 mg/kg/day) onwards in either 6- or 23-day studies in rats, and by Chan, who also reported an impairment of torsional strength with 10 mg/kg/day [8]; and were also in consonance with the impairment of both bone mineral content and Ca absorption observed by axter giving high doses of EHDP during 4-6 weeks to chicks [41].

As both geometrical and material properties contribute to determine the me- chanical performance at the organ level [12,29,33], both positive and negative cor- relations shown in Fig. 3 should be interpreted in relation to the nature of strength (biphasic) and stiffness (monophasic) curves in Fig. 1. These suggest that either at low or high doses the effects on material properties were those predominant in de- termining diaphyseal strength, in agreement with Currey [33], though they did not affect diaphyseal rigidity. In this regard, the relative irrelevance of mass changes (volume, wall/lumen ratio) should be referred to (a) the lack of new mineralization demonstrated from 4.5 mglkglday onwards [ 161 which can potentially lea cumulation of mechanically inoperant, uncalcified osteoid as has been shown after EHDP treatment [8,41] and/or (b) the lack of variation of the sectional moment of inertia (Fig. 5) probably derived from blockage of osteoblast-osteoclast communi- cation. In fact, the sectional moment of inertia (Ix, related in this case to the hori- zontal axis, that is, to that perpendicular to the direction of the acting force) is more closely related to architectural support than the volume or wall/lumen ratio, since these latter merely reflect bone mass accumulation [12,13,29]. The differential ef- fects of treatment on internal diameters b (largely reduced) and h (slightly en- larged) in rel,t.,.. Sy ti_Xirl ..-. 9 inn +I* av+amnt diameters B and N (Fig. 4) and their particular influ- ence on the formulae for calculations of the geometrical variables (a) explain the apparently paradoxical constancy of Ix in spite of the positive changes of volume

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and wall/lumen ratio at high doses and (b) show how the resulting over-normal amount of bone mass tended to be inadequately arranged throughout the bone sec- tion from an architectural point of view. This latter argument is especially impor- tant considering that the assayed bones were loaded following the sense of action of the most common (physiological) challenging stresses, so that Ix represented both a geometrical and mechanical parameter for our animals.

In brief, our data show that APD effects on normal rat long bone biomechanics, apart from being dose-time dependent. are biphasic and asymmetric: at low doses they are beneficial to diaphyseal strength because of an increment in material quali- ty derived from improved calcification; and at high doses they are detrimental to diaphyseal strength and stiffness because of a reduction in material quality derived from impaired calcification and inadequately compensated by an architectural and mechanically superfluous increment of uncalcified bone matrix.

Acknowledgements

This work was supported by grants (P.I.D.‘s Nrs. 3-019800/85 and 3-092400/88) from the Consejo National de Investigaciones Cientificas y T&cnicas (WNICET). J.L.F. is a Career Investigator of the Consejo de Investigaciones de la Universidad National de Rosario (CIUNR). Technical assistance from Mr F. Fernandez and Mrs N. Meoli, M.I. Zanor, G.R. Brunori and I.M. Menoyo is gratefully acknowl- edged. APD was kindly provided by Dr Gadof Laboratories, Buenos Aires.

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