Differential Action of the Bisphosphonates

(3-Amino-i -Hydroxypropylidene) -1,1 -Bisphosphonate

(APD) and Disodium DichloromethylideneBisphosphonate (C12MDP) on Rat Macrophage-mediatedBone Resorption In VitroPIETER H. REITSMA, STEVENL. TEITELBAUM, OLAV L. M. BIJVOET, and

ARNOLDJ. KAHN, Department of Clinical Endocrinology, University Hospital,Leiden, The Netherlands; Department of Pathology and LaboratoryMedicine, and the Division of Bone and Mineral Metabolism, The JewishHospital of St. Louis, and Washington University Schools of Medicineand Dental Medicine, St. Louis, Missouri 63110

A B S T R A C T The bisphosphonates (3-amino-1-hy-droxypropylidene)-1,1-bisphosphonate (APD) and di-sodium dichloromethylidene bisphosphonate (Cl2MDP)effectively inhibit the accelerated bone resorption as-sociated with some skeletal disorders, e.g., Paget's dis-ease. However, it has not been established whetherthese compounds exert their inhibitory effect by ren-dering the bone mineral more resistant to degradation,by diminishing the activity of resorbing cells, orthrough some combination of both activities. In thisstudy, we have tested these possibilities using an invitro resorption assay system consisting of elicited ratperitoneal macrophages co-cultured with particles of45Ca-labeled, devitalized rat bone. This assay systempermits the quantitative assessment of the action ofAPD and Cl2MDP on the two major phases of boneresorption (cell-substrate attachment and osteolysis)under circumstances where the drugs are present con-tinuously or, most importantly for the issues in ques-tion, after the separate pretreatment of the particlesor the resorbing cells.

Our data indicate that (a) Both APD and C12MDPat concentrations 25 X 106 Mdiminish macrophage-mediated 45Ca release (i.e., bone resorption) in a log

Received for publication 22 February 1982 and in revisedform 9 July 1982.

Address correspondence to Dr. Pieter H. Reitsma, Wash-ington University School of Dental Medicine, St. Louis, MO63110.

dose-dependent fashion. (b) A 10-min pretreatment ofbone particles with either bisphosphonate (P-C-P) sim-ilarly inhibits resorptive activity, but is most pro-nounced with Cl2MDP. However, only APD is effec-tive in reducing resorption when cells are preincu-bated (for 24 h) with P-C-P. (c) In cultures containingboth labeled and unlabeled bone, significant inhibitionoccurs only when the labeled particles are coated withP-C-P (indicating that the action of P-C-P-treatedbone is highly localized). (d) P-C-P does not diminishcell-bone particle attachment, an essential step in theresorptive process. On the other hand, delaying theaddition of P-C-P until after cell-bone attachment iscompleted significantly reduces the resorption-inhib-iting effect of these compounds. (e) C12MDPreducesculture DNA content in proportion to its inhibitoryeffect on resorption, and both the inhibitory and cy-totoxic actions of this P-C-P are dependent upon thepresence of bone. On the other hand, APD is cytotoxiconly at very high concentrations (10-4 M), acts inde-pendently of the presence of bone, and inhibits re-sorption without killing cells.

Weconclude that the mechanisms of action of APDand C12MDPare markedly different. C12MDPis a po-tent cytotoxin in the presence of bone and apparentlyexerts its inhibitory effect in this manner. APDis non-cytotoxic at levels adequate to suppress resorption and,therefore, must inhibit macrophage activity by someother mechanism. Neither P-C-P appears to limit re-sorption by decreasing the solubility of mineralizedbone matrix.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/82/11/0927/07 $1.00Volume 70 November 1982 927-933

927

INTRODUCTION

Bisphosphonates (P-C-P)' are nonbiodegradable ana-logues of pyrophosphate (PPi) with a high binding af-finity for hydroxylapatite crystals (1). Administrationof P-C-P to experimental animals rapidly curtails boneresorption (2-4), and it is for this reason that thesecompounds are used in the treatment of clinical dis-orders characterized by increased bone degradation.For example, both disodium (3-amino-l-hydroxypro-pylidene)-1,1-bisphosphonate (APD) and disodiumdichloromethylidene bisphosphonate (Cl2MDP) areeffective in decreasing bone resorption in Paget's dis-ease and in the osteolytic lesions associated with neo-plastic bone disease (5-8). Interestingly, APD effec-tively decreases bone resorption in vivo at much lowerdoses than does C12MDP. There is no clear explanationfor this difference in potency, but it may relate to theobserved effects of APD on the mononuclear phago-cyte system (9). Cells of the mononuclear phagocytesystem (MPS) have the potential to regulate osteoclastactivity (e.g., by producing prostaglandin E2) and APDmight alter this aspect of MPSfunction.

The basis for the resorption-inhibiting effects ofP-C-P remains unknown, but given the binding affin-ity of these compounds for hydroxylapatite crystals,they may well act subsequent to accumulation on bonemineral surfaces. Once present on such surfaces, theymay exert their inhibitory effect by (a) directly de-creasing mineral solubility and ultimately the resorb-ability of bone matrix, (b) inhibiting the attachmentof osteoclasts and other potential resorbing cells (mac-rophages, tumor cells) to bone surfaces, or (c) dimin-ishing the resorption-related and/or general metabolicactivity of osteoclasts and other osteolytic cells. Alter-natively, circulating P-C-P may act directly on cellsto block cell-bone attachment or resorption, withoutprior binding to mineral surface.

At present, there is little information regarding therelative biological significance of each of these possiblemechanisms of P-C-P inhibition. It is known, for ex-ample, that P-C-P will inhibit the growth and disso-lution of hydroxylapatite crystals in vitro (10, 11). Inaddition, P-C-P have been shown to modify the met-abolic activity of several different cell types grown inculture (12, 13). However, in none of these cases havebone matrix (or mineral) and a cell with osteolyticpotential been separately treated with P-C-P and thencombined in such a way as to assess the subsequent

I Abbreviations used in this paper: APD, (3-amino-1-hy-droxypropylidene)-1,1-bisphosphonate; Cl2MDP, disodiumdichloromethylidene bisphosphonate; a-MEM, Eagle's min-imal essential medium; M0, adherent cell; MPS, mononu-clear phagocyte system; P-C-P, bisphosphonate(s); PPi, in-organic pyrophosphate.

action of these compounds on the resorptive process.In this study, we have explored the mechanism of

action of C12MDPand APD using a recently devel-oped, macrophage-based assay system for bone re-sorption (14). With this system, it is possible to inde-pendently treat resorbing cells (elicited peritonealmacrophages) and bone mineral (devitalized rat boneparticles) with P-C-P and to quantify the results ofsuch treatment on the ability of the macrophages tobind and resorb bone. Our data indicate that APDandC12MDPdo not alter the attachment of cells to bonebut do inhibit macrophage-mediated resorption in adose-dependent fashion. In the case of C12MDP, thisinhibition is bone mineral dependent, occurs primarilysubsequent to the accumulation of P-C-P on the bonecrystal surface, and appears to be a manifestation ofthe cytotoxicity of this P-C-P. In the case of APD, onthe other hand, the P-C-P is cytotoxic only at highconcentration, acts independently of the presence ofbone and inhibits resorption, at least in part, by amechanism not yet defined but probably dependentupon the direct action of APD on cells. NeitherP-C-P appears to affect resorption by reducing the sol-ubility of the mineral phase of bone.

METHODS

Binding and resorption assays are performed as described(14) and are summarized as follows: Elicited peritoneal ad-herent cells (M0) were obtained from rats (Sprague-Dawley,±150 g) 3 d after a peritoneal injection with 10% Brewer'sthioglycollate medium. The exudate cells were allowed toattach for 1 h to the wells of Costar dishes (5 X 105 cells/16-mmwell) in 0.5 ml modified Eagle's minimal essential me-dium (a-MEM) buffered to pH 7.4 with 3N-morpholinopro-panesulfonic acid. The cultures were rinsed, and a-10 me-dium (bicarbonate-buffered a-MEM supplermiented with10% fetal calf serum) was added to each well. The plateswere incubated at 370C in humidified air and 5% CO2 foran additional 18-23 h, and then vigorously rinsed to removenonadherent cells. The remaining adherent cells were 95-98% macrophages as assessed by phagocytosis and nonspe-cific esterase staining.

Particles (<25 um) of devitalized rat bone, labeled in vivowith 45Ca, were suspended in a-10 medium and were addedto the macrophage cultures to yield a final concentration of0.115 mg/well. Binding of the bone particles to the cells wasassessed after 2 h of incubation by recovering the nonad-herent particles from the culture surface and determiningthe isotopic content of the adherent and nonadherent par-ticle fractions. Bone resorption (expressed as net percentageof 45Ca release) was routinely determined after 96 h incu-bation from the percentage of 45Ca released into the mediumas a consequence of macrophage activity, and was correctedfor physicochemical exchange of 45Ca that occurs when boneis incubated in the absence of cells. The results of both bind-ing and resorption assays were derived from six replicatecultures for each variable and are presented as mean±SEM.Experiments were generally repeated two to three timesbefore the findings were accepted as valid.

The bisphosphonates APD and Cl2MDP, and PPi were

928 P. H. Reitsma, S. L. Teitelbaum, 0. L. M. Bijvoet, and A. J. Kahn

dissolved in 0.9% saline and diluted in a-10 medium to thedesired concentrations before addition to the cultures. Al-ternatively, bone particles are pretreated by incubating themfor 10 min in solutions of P-C-P, PP1, or phosphate buffer,resuspended in a-10 medium and added to the cultures.Pretreatment of the M0 was achieved by incubating thecells for 24 h in P-C-P contained in a-10 medium, and thenrinsing and adding labeled bone to the cultures. Cell numberand viability were indirectly assessed by measuring the DNAcontent of the M0 population using the ethidium bromidetechnique described by Karsten and Wollenberger (15).

RESULTS

APDand C12MDP, when present throughout the entireassay period, reduced net 45Ca release from the boneparticles in a log dose-dependent fashion. In the rep-resentative experiment shown in Fig. 1, significant in-hibition of resorption was achieved by both com-pounds at 5 X 106 M (P < 0.01). The molar potencyof the two P-C-P was equal. On the other hand, thepassive efflux of 45Ca from bone particles incubatedin the absence of cells, which accounted for -20% ofthe radioactivity in the culture medium, was not al-tered by either APD or Cl2MDP (data not shown).

Inhibition of 45Ca release also occurred when par-ticles were preincubated for 10 min in solutions con-taining APDor C12MDPbefore being introduced intocultures. Using this approach, C12MDPwas more ef-fective, on a molar basis, than APD, whereas pretreat-ment in control solutions of PPi or phosphate bufferwas without effect (Fig. 2). When compared with its

50

40Lu

,, 30

Ug 20 -

LUiz

10 0 Control* APDO C12MDP

10-7 10-6 10-5 10-4CONCENTRATION(M)

FIGURE 1 P-C-P inhibition of 45Ca release by M0 from de-vitalized 45Ca-labeled bone particles after 96 h incubation.APDand Cl2MDP were continuously present at the concen-trations indicated. Net percent 45Ca release was calculatedby subtracting the 45Ca release from cultures with bone par-ticles only (passive efflux) from the 45Ca release in the cul-tures containing MOand bone particles. All values representmean±SEMfrom six determinations.

LU

LU40-LU

20-

I-)LUz

Control 4 O M10-4,1t4 A10-5A 104M.P04 PPi C12MDP APD

FIGURE 2 Effect of 10-min preincubation of devitalized45Ca-labeled bone particles with APD, CI2MDP, PP1 or P04,on 45Ca release by M0. All values represent mean±SEMfrom six determinations.

continuous presence in cultures, the dose-responsecurve for preincubation of bone particles with Cl2MDPwas shifted to the right by about half an order of mag-nitude (Fig. 3). In marked contrast, only APD waseffective in reducing macrophage-mediated resorptionunder circumstances where the cells were preincu-bated with P-C-P for 24 h (Table I).

Additional evidence for a direct involvement ofbone surfaces in mediating the inhibitory action ofP-C-P was obtained from experiments in which 45Ca-labeled and unlabeled bone were introduced concur-rently into replicate M0-containing cultures. In some

60 - Controls

50 \LU

Lu 40-

0 Prea~~~~~~~~ncubotions

tW 20 Continusz

10 \

I10-6 lo-5 lo-4 10-3

CONCENTRATION(M)FIGURE 3 Comparison of bone particle pretreatment withcontinuous exposure to Cl2MDP on macrophage-mediatedresorption. Additional controls were bone particles prein-cubated with PO4-buffer (2 X 10' M, open triangles) andPP; (lo-4 M, closed triangles). Note the relatively small dif-ference in dose-response between the two modes of intro-ducing P-C-P. All values represent the mean±SEM for sixreplicate cultures.

Action of Bisphosphonates on Bone Resorption 929

TABLE IEffect of Cl2MDP and APDon MO-mediated Bone Resorption

when Mb Were Preincubated with P-C-P for 24 hbefore the Introduction of Bone

Control Net 'Ca release

39.5±1.95 X 10-5 MAPD 17.3±3.9°5 X 10-6 MAPD 39.5±1.75 X 10-7 MAPD 37.9±1.05 X 10-5 MC12MDP 43.7±2.45 X 10-6 MC12MDP 41.1±1.05 X 1o-7 MC12MDP 40.6±1.6

All values are means±SEM from six determinations.' Significantly different from control, P < 0.01.

instances, the labeled particles were pretreated withP-C-P before being added to the wells; in other cases,it was the unlabeled bone that was coated withP-C-P. If P-C-P act primarily by their presence at thecell-bone interface, then only those wells containinglabeled bone and P-C-P should show evidence of re-duced resorptive activity. In the experiment depictedin Fig. 4, the decrements in apparent resorptive ac-tivity were only significant in cultures containing la-beled bone pretreated with P-C-P.

We have previously demonstrated that binding ofbone by MOis a prerequisite for resorption and thata lag period of -8 h exists between the initiation of

14-

1212

10

LU

U L U L U L |

PP C12MDP APD

FIGURE 4 The co-culture of labeled and unlabeled bone"coated" with C12MDPor APD. In some instances the la-beled bone (L) was pretreated (10 min) with 1i-0 MC12MDPor APD; in other cases the unlabeled bone (U) was pre-treated. Notice that significant (*) inhibition of resorption(45Ca release) was evident only in those instances where thelabeled particles were coated with P-C-P (P < 0.01, C12MDP;P < 0.001, APD). All values represent the mean±SEMfromsix determinations.

TABLE IIEffect of Cl2MDP and APDon the 2-h Attachment

of Bone Particles to Cells

Control Particles bound

80.5±0.95 X 10-5 M APD 81.0±0.75 X 10-7 M APD 81.8±0.95 X 10-5 M C12MDP 82.5±0.45 X 10-7 M C12MDP 83.2±0.7

All values are means±SEM from six determinations.

binding and the onset of osteolysis (14, and unpub-lished observations). Table II shows that neither com-pound interferes with the binding of bone by the cells.However, deferring the introduction of P-C-P untilafter maximum cell-particle binding is achieved andbone degradation begins, significantly reduces the re-sorption-inhibiting effect of these compounds (Fig. 5).This effect was most notable with C12MDPwhere an8-h delay totally eliminated the suppressive effect ofthis P-C-P. APD, on the other hand, continued to be

50 C12MDP,5 x10-6M

40

30

20-

Lu10

30APD, 5 10-MM

30z

20-

10

It0 t4 t8 Control

HOUROF ADDITION

FIGURE 5 Effect of delayed addition of APD and C12MDPon the inhibition of 45Ca release by MOfrom devitalizedbone particles after 96 h incubation. Note that in all cases,delaying the introduction of P-C-P renders the compoundssignificantly less inhibitory (P < 0.01 to P < 0.001), but thatthe effect is most pronounced with C12MDP. All values rep-resent the mean±SEM from six determinations.

930 P. H. Reitsma, S. L. Teitelbaum, 0. L. M. Bijvoet, and A. J. Kahn

strongly inhibitory even when introduced after theparticle binding phase was completed.

Chambers (18) recently reported that high concen-trations of C12MDPare cytotoxic for murine macro-phages. This cytotoxicity was also evident in rat peri-toneal cells and likely accounts for all the inhibitionof MO-mediated resorption exhibited by this P-C-Pin vitro. For example, as can be seen in Fig. 6, incultures containing bone particles and treated withC12MDP, for the 96-h assay period, there is a nearproportionality between the degree of cytotoxicity(loss of DNA) and the extent of the inhibition. Wenoted earlier the effectiveness of pretreating bone par-ticles with Cl2MDP in suppressing resorption (Fig. 2).The critical importance of the bone particles in con-tributing to the action of Cl2MDP was further docu-mented by experiments in which cytotoxicity wasshown to vary as a function of both the amount andparticle size of bone added to the cultures. From theexperiment depicted in Fig. 7, it can be seen that thedecrement in the amount of DNAincreases with in-creasing amounts of bone, and that smaller bone par-ticles are approximately an order of magnitude moreeffective in enhancing cytotoxicity than are largerones.

The relationship between cytotoxicity and the in-hibition of resorption was much less certain with re-gard to APD and C12MDP. For example, in contrastto C12MDP, cultures treated continuously with APDexhibit relatively greater inhibition of resorption thanloss of cells (Fig. 6). Moreover, and again in distinction

LUL/,

LJLJu0_

_Utu -;

a, >

14cZ2

-4,

z

RELATIVE DNACONTENT(X Reduction vs. Controls)

FIGURE 6 Relationship between the inhibition of 45Ca re-lease and the reduction in DNAcontent of the M0 popu-lation in incubations containing bone particles. The concen-trations of APD (circles) and Cl2MDP (squares) were 10-6M (open symbols), 10-5 M (striped symbols), and 10-4 M(filled symbols). Notice that with APD, the inhibition of re-sorption is greater than would be expected on the basis ofcytotoxicity (loss of DNA) alone. All values represent themean±SEMfrom six determinations.

2.01

u-Iui

z

0~

1.5

1.0

0.5

0 0.01 0.1 1.0BONE/mg PER WELL

FIGURE 7 CI2MDP cytotoxicity as a function of the amountand particle size of bone added to macrophage cultures. (Inthis figure 0 denotes diameter of bone particles.) Note thatincreasing the amount of bone leads to increased cytotoxicity(measured as the loss of DNAper well) and that smaller boneparticles are more effective in enhancing cytotoxic activitythan are larger ones. Each value represents the mean±SEMfrom six replicate cultures.

to Cl2MDP, the degree of cell death in APD-treatedcultures was independent of the mode of presentationof the P-C-P and whether or not bone was present inthe wells (Table III). Finally, and perhaps most im-portantly, APD showed significant inhibition of re-sorption at concentrations lower than those needed to

TABLE IIIEffect of APDand Cl2MDP on DNAContent of Cultures after

96-h Incubation

DNA/well, sg

Control 2.76±0.07

Continuous Preincubation, Preincubationexposure, with with bone cells (no bonebone particles particles particles)

DAP10-6 M 3.02±0.09 2.96±0.04 2.85±0.10i0-5 M 2.80±0.05 2.74±0.06 2.64±0.0710-4 M 2.18±0.07t 2.21±0.021 1.96±0.10t

C12MDP10-6 M 2.20±0.041 2.53±0.06* -10-5 M 1.03±0.101 1.63±0.061 t10-4 M 0.71±0.10t 0.78±0.071 t -

All values are means±SEMfrom six determinations.Significantly different from control P < 0.05.

I Significantly different from control, P < 0.001.

Action of Bisphosphonates on Bone Resorption 931

affect cell viability (loss of DNA; c.f., Fig. 6), and witha different time course of activity (Fig. 8).

DISCUSSION

Wementioned previously that P-C-P bind to and limitthe in vitro solubility of hydroxylapatite (bone) crystals(1, 10, 11). It follows from this that P-C-P may inhibitbone resorption by directly increasing the resistanceof the mineral phase of bone to degradation (16). How-ever, the inhibition of resorption by P-C-P is also as-sociated with dramatic changes in both osteoclast mor-phology and in the size of the osteoclast population(2-4, 17), indicating that these compounds exert a pro-found effect upon resorbing cells as well as upon thebone mineral.

To gain further insight into the relative importanceof P-C-P action on mineralized bone matrix and re-sorbing cells, we have assessed the effect of these com-pounds using an in vitro assay system that permits theindependent treatment of the cellular and matricalcomponents of bone resorption. Specifically, w'e havedetermined the binding and resorption of isotopicallylabeled rat bone particles by elicited rat macrophagesunder conditions where the drugs were present con-tinuously and following the preincubation of the cellsor bone particles with P-C-P.

The suppression of 45Ca release in the continuouspresence of Cl2MDP or APDdocuments the inhibitoryeffect of P-C-P on bone resorption in the macrophage-resorption system. Moreover, the lack of change in thepassive movement of 45Ca in the presence of eithercompound argues th'at this inhibition cannot be ex-plained simply by crystal stabilization. Similarly, the

40

30-uJ

320

t10uz

,2z

effect of P-C-P on resorption does not appear to bedue to a diminution in the' avidity with which resorb-ing cells bind the bone particles, a step that we havepreviously shown to be essential in the resorptive pro-cess (14).

On the other hand, our data do indicate that thebinding of P-C-P to bone mineral very likely plays animportant role in the skeletal action of these com-pounds, at least with regard to Cl2MDP. For example,pretreatment of 45Ca-labeled bone particles withC12MDPsuppresses subsequent resorptive activity al-most as effectively as having the P-C-P present con-tinuously in the culture medium. In addition, the ob-servation that the inhibitory action of P-C-P is reducedby delaying their introduction into the culture suggeststhat the compounds lose at least some of their effec-tiveness if they are prevented from accumulating oncrystals' surfaces until cell-matrix attachment is com-pleted and resorption initiated. This observation isparticularly striking in the case of Cl2MDP, where an8-h delay entirely mitigates its inhibitory action. Fi-nally, the data from the experiment utilizing treatedunlabeled and labeled bone further establishes thatmineral-bound P-C-P can directly suppress resorptionwithout prior release of P-C-P from the bone surfaceor of some secondary effector substance (monokine)from macrophages.

The proportional diminution in the DNA contentof adherent macrophage populations and resorptiveactivity indicates that cytotoxicity is largely respon-sible for the inhibitory effect of C12MDPon bone re-sorption, as has been previously suggested (18). Thiscytotoxicity is dependent upon the presence of boneand, as we have shown here, increases when additional

TIME (h )FIGURE 8 Time course of the inhibition of resorption by 5 X lo-5 MC12MDPand APD. Notethat both P-C-P show marginal inhibitory activity within the first 24 h of culture and significantinhibition by 48 h. C12MDPis clearly more potent than APDand essentially curtails all resorptiveactivity after the 1st d of incubation. In this experiment only Cl2MDP proved cytotoxic, reducingculture DNAvalues by 72 h to 1.38 jug/well vs. 2.13±0.13 for controls and 2.05±0.15 for APD-treated cultures. Each value represents the mean±SEMfrom six replicate cultures.

932 P. H. Reitsma, S. L. Teitelbaum, 0. L. M. Bijvoet, and A. J. Kahn

bone particles are added to the cultures. We notedabove the importance of bone surfaces in "delivering"P-C-P. This aspect of P-C-P action is reinforced by theobservation that smaller bone particles, with their sub-stantial surface-to-volume ratio, are significantly moreeffective in enhancing cytotoxicity than are larger par-ticles (even if the latter are present in much greateramounts).

In contrast to C12MDP, APDdoes not appear to in-hibit macrophage-mediated resorption solely by kill-ing the resorbing cells. For example, there is a poorcorrespondence between the loss of adherent macro-phages and the inhibition of resorption in culturestreated with APD, and, in fact, significant inhibitionis usually observed in the absence of a demonstrablecytotoxicity. Moreover, when cytotoxicity is encoun-tered, it is at very high concentrations of APD (1loM) and is independent of both the mode of introduc-tion of the P-C-P and whether or not bone is presentin the cultures.

APDand C12MDPdo not behave identically in vivo(9) and other differences in the mechanisms of actionof these compounds are evident in the present in vitrostudy. C12MDPappears to work as a cytotoxin in thepresence of bone, and this seems adequate to accountfor the diminution in macrophage-mediated resorp-tion reported above and, perhaps, in animals treatedwith this P-C-P. APD, on the other hand, suppressesresorption without killing the resorbing cells and with-out the intervention by bone. This latter effect is, per-haps, most notable in the delayed introduction exper-iments, where APDcontinues to exhibit significant in-hibitory action even after the completion of cell-bonebinding. Thus, while the precise mechanism of actionof APD remains unknown, the fact that it can act di-rectly on sensitive cells may account for the relativelygreater in vivo potency exhibited by this compoundwhen compared to other P-C-P, e.g., C12MDP andEHDP(1-hydroxyethylidene-l,1-bisphosphonate).

ACKNOWLEDGMENTSThe authors wish to acknowledge the expert technical assis-tance of Jenny Sadow and Lianne van der Wee-Pals. APDand Cl2MDP were kindly donated by Henkel KGaA, Dus-seldorf, West Germany.

This work was supported, in part, by National Institutesof Health grant DE-05413 and DE-04629 and by the Foun-dation for Medical Research (FUNGO), which is subsidizedby the Netherlands Organization for the Advancement ofPure Research (ZWO).

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