Effect of polyvinyl phosphonates and ethane hydroxy diphosphonate on mineralization of ectopic bone

Chem -Bml Interaetmns, 48 (1984) 339--347 Elsevier Scientific Pubhshers Ireland Ltd.

339

E F F E C T OF POLYVINYL PHOSPHONATES AND ETHANE HYDROXY DIPHOSPHONATE ON I~INERALIZATION OF ECTOPIC BONE

N C RATH*, S DIMITRIJEVICH and M ANBAR

Department of Bmphys~cal Sctences, State Unwers~ty of New York at Buffalo, Buffalo, NY14214 (USA) (Received November 22rid, 1982) (Revlsmn received September 7th, 1983) (Accepted September 17th, 1983)

SUMMARY

The effects of polymeric polyphosphonate and of ethane hydroxydlphosphonate on the development of ectopyc bone were compared at different stages of bone development. The diphosphonate affected bone development at different stages, as reflected by alkalme phosphatase and aryl sulphatase activities, as well as by the creation of calcium ions by bone and on its readiness to undergo lsotopm exchange. The polymerm polyphosphonate (mol. wt., 3500--8000), on the other hand, did not exhibit any of these effects, although it did inhibit the activity of the enzymes m vitro to an extent comparable with that of the dlphosphonate. The results corroborate the assumphon that the effects of polymeric polyphosphonates on hard tissues are confined to the extracellular space while the effects of dlphosphonates on bone development are due to mtracellular activity and not to sequestermg of extracellular calcium.

Key words Polyphosphonate -- Dlsphosphonate -- Ectopic bone -- Minerallzatlon INTRODUCTION

Dlphosphonates have gamed attention In recent years in the treatment of metabohc disorders involving calcification and bone resorption [1--5]. These compounds have high affinity for calcified surfaces and prevent both for-

*Present address Internattonal Genetms Engineering, Inc 1701 Colorado Avenue, Santa Monma, CA 90404, U S A Abbreviations EHDP, ethane hydroxy 1,1-dlphosphonate, PVP, polyvmyl phosphonate, VPD, vinyl phosphonyl dichloride

340 mat:on and dissolution of h y d r o x y a p a t l t e m vitro. The effectiveness of these compounds m vlvo is due to the presence of C-P bonds, whmh :mparts resistance to their metabohc degradatmn by endogenous phosphatases [2--7]. Phosphonates may have biologmal act:vlty m areas different from calcffmatlon or decalcfficatmn. Because of their low molecular weight, d:phosphonates have ready access into cells and interfere with some processess of carbohydrate metabohsm such as glycolysls [8,9]. Prehmmary studms of certmn polymer:c polyvlnyl phosphonates (PVP) by Anbar et al. lndmate that these high molecular weight compounds, which share m a n y c o m m o n physmochemmal propert:es with dlphosphonates and have slgmficant antmarms activity [10,11], do not exhib:t mtracellular effects, since their p o l y m e n c and p o l y a m o m c nature would not permit the:r easy access into cells. Further, the polymer:c nature of these compounds permits the m o d i f m a t m n of their pharmacological properties by lntroduct:on of other functmnal groups such as fluoride [12], whmh could be distinctly advantageous m preventing bone loss or :n facilitating mmerahzation. These conslderatmns prompted us to investigate the effect of PVP on the mmerahzat:on of bone. The studms reported here have been done using a model of ectop:c bone reduced by subcutaneous :mplantation of decalcified bone matrLx [13--15]. The specific advantage of th:s system :s ~ts temporally distract developmental phases, wh:ch perm:ts phys:ologmal and pharmacologmal mampulatlons w i t h o u t mterfenng with the preceding sequence [15,16]. In this model subcutaneous :mplantation of decalcff:ed bone matrix m rats causes an inflammatory response on day 0 culminating :n local prohferat:on of putative chondroblasts to reach a peak on day 3. These cells subsequently differentiate to chondrocytes forming cartilage by day 7, wh:ch undergoes chondrolysis subsequently. A simultaneous vascula invasmn and cell prohferation induces osteogenes:s, forming a new bone by day 11--12 of the lmplantat:on of the matr:x. The new bone mmerahzes and remodels, forming a complete hematopoeitm ossicle by 3 weeks [14]. The comparative effects of PVP and ethane h y d r o x y 1,1-d:phosphonate (EHDP) on osteogenes:s and mmerallzatmn of ectopm bone were investigated and are presented m this paper. MATERIALS AND METHODS

Synthes,s of PVP Vinyl phosphonyl dmhlor:de (VPD) was prepared according to a modff:ed earher procedure [11] and pur:fmd by double fractlonatlon. Ten grams of VPD dissolved m 50 ml hexane containing 0.5 g of Azob:s 2-methyl proplomtnle were p o l y m e n z e d m solution by heating under reflux m a dD' N2 environment. Following polymerization, the hexane was removed by blowing with N2 and the residue was dissolved m water followed by low pressure evaporation of the water, and the process was repeated several times, ensunng total removal of the HC1 that was produced during the polymerization. The residue was finally dissolved m water, neutrahzed with

341 0 2 N NaOH, filtered through cehte and dialyzed against water with a Spectrapor 6 ® membrane with mol. wt. cut-off of 3500. The retentate was further transferred to Spectrapor @ membrane with mol. wt. cut-off of 8000 and dialyzed against water. The dlalysate was evaporated and used as PVP with mol. wt. 3500--8000. EHDP-~sodmm (mol. wt., 250) was donated by Dr. M. Francis of Procter and Gamble Company, Cincinnati, OH.

Induction of bone and 4SCa-mcorporatton in vivo Demmerahzed bone matrix prepared from rat diaphyses were implanted on bilateral sites of a n t e n o r thoracm region of 50--80 g male rats of the Long Evans strain on day 0. In one experimental group the injection of PVP or EHDP was Initiated on day 7, at the cartilage phase, whereas the other groups were injected with the drugs from day 10 onwards until autopsy on day 15. The drugs were injected m salme solution mtraperitoneally daily at a dose of 10 or 100 mg/kg, respectively, whereas the control rats received only saline. The autopsms were performed 20 h after the last injection. Two hours p n o r to autopsy, 4SCaC12 (spec. act., 18.3 mCi/mg, ICN) was Injected ;ntrapentoneally at a dose of 1 pC1/g body wt. Prior to sacnfice, blood was collected from individual rats by cardiac puncture to determine the serum level of radioactivity, and the concentrations of free calcium and of inorganic phosphorus. Calcium concentration was determined with Perkln Elmer Atomm absorption spectrometer according to bulletin No. 555 and Inorganic phosphorus according to Chen et al. [17]. At autopsy, the ossicles were weighed and processed for determination of alkaline phosphatase, aryl sulfatase, as well as 4SCa-mcorporatlon, following earher procedures [13,18,19]. The ossicles were homogenized m Tris--HC1 buffer (pH 7.4), centrifuged and the supernatant saved for enzyme determinations using hydrolysis of p-nltrophenyl phosphate and p-mtrocatechol sulfate at pH 9.3 and 5.5, respectively [19,20]. The residue was equilibrated with 0.1 M CaC12, Tris--HC1 (pH 7.4) for 30 mln with constant stirring, then the samples were centrifuged and the supernatant was discarded. Following two successive washings and centrlfugatmn with Trls--HC1 buffer (pH 7.4), the residue was dissolved overnight in 0.5 M HC1 and centrifuged. The incorporated 4SCa was determined by counting an aliquot o f the 10 000 × g supernatant. Total concentration ot calcium and inorganic phosphate m this extract were determined as described above. The results were expressed per milligram weight of the tissues and normahzed to serum level of radloactiwty using the following formula: cpm/mg tissue × 104 cpm/0 1 ml serum Portions of day 14 control and treated OSSlCles were decalcified and processed according to routine hmtologmal procedures and examined under a hght mmroscope to determine their developmental status.

342

In vitro effect o f P V P and EHDP on 4SCa-mcorporatmn Since it is k n o w n t h at the phosphonat es may exert thmr effect by adsorption to mineralized surface, an additional e x p e r i m e n t was designed to study their effect by incubating the ossmles with EHDP or PVP for 30 man followed by testing their ability to incorporate 4SCa in vitro. For this purpose 4-week-old ossicles f r om 8 rats were pooled and cut m t o wedges of 20--25 mg and r a n d o m l y allocated to three separate groups, four Indwldual experiments m each group. Each Individual e x p e r i m e n t was carried out with r a ndom l y selected 5--7 wedges and incubated m a calcifying buffer containmg 85 mM NaC1, 15 mM KC1, 10 mM NaH2CO3, 50 mM Tns, 1.6 mM KH2PO4 and 2.2 mM CaCI: (pH 7.4) at 37°C according to Anderson and Sajdera [19--21]. PVP and EHDP were added at a c o n c e n t r a t m n of 1 mg/ml and the wedges were incubated for 30 mm m a shaking water bath The tissues were then briefly nnsed with buffer and transferred to fresh buffer containing 1 t~CI/ml of 4SCaCl~ to be furt her incubated for 2 h. The tassues were t hen washed for 10 m m by constant st~rrmg wath a magnetm bar m excess of buffer, blot dried, weighed and h o m o g e m z e d m a buffer of 50 mM Tns--HC1 (pH 7.4) containing 0.1 M CaC12 and starred for 30 m m and centnfuged. An a h q u o t whmh represents exchangeable, non-specifmally b o u n d calcium was c o u n t e d for radmactwlty. The precipitate was further washed twine with buffer and e x t r a c t e d overmght with 0.5 M HC1 and the radmactivity was c ounted, to represent incorporation of 4SCa anto minerals. The results were stat~stmally evaluated using Student's t-test. RESULTS

The results of the studms are presented In Tables I - IV. Neither EHDP nor PVP had any effect on plasma levels of calcmm and morgamc phosphorus (Table I). However, the t r e a t m e n t with EHDP caused a slgmfmant reduction in the i ncor por at i on o f 45Ca into ectoplc ossmles, an effect whmh was more TABLE I CALCIUM AND INORGANIC PHOSPHORUS LEVELS OSSICLES OF R A T S T R E A T E D WITH PVP AND EHDP

OF S E R U M A N D D A Y

15

T h e r a t s w e r e t r e a t e d w i t h t h e d r u g s f r o m d a y 7 - - 1 3 M e a n ~alue fok c a l c m m a n d phosp h o r u s m s e r u m was o b t a i n e d f r o m 4 rats m e a c h g~oup w h e r e a s t h e m e a n values fo~ o s s m l e s w e r e o b t a i n e d f r o m 7 - - 8 s a m p l e s o b t a i n e d f r o m 4 rats Treatment

Serum Calemm (mg/+_S E M )

Control PVP EHDP

811+048 8 52_+195 8 2 9 - + 0 58

Osslcle Phosphorus ( m g / 1 0 0 ml

Calemm (umol/mg

Phosphotus (umol/mg

_+SEM)

_+SEM)

_+SEM)

523+012 5 6 5 - / - - 0 13 4 8 9 + _ 0 38

086_+009 1 01 +_0 08 0 16-+0 0006

057_+005 0 64 -+0 06 0 04-+0 002

P Ca

1 151 1 1 58 1 3 90

343 TABLE II EFFECT OF PVP AND EHDP ON METABOLIC CHEMISTRY OF DAY 15 OSSICLES (mean+SEM) Expt 2 The rats were treated with the drugs from day 7--14 Expt 3 The treatments were from day 10--14 The results are mean of 7--8 ossmles obtained from 4 rats in each experimental group The results were evaluated m reference to control values *P < 0 01, **P < 0 O01,***not stgmflcant Expt no

Treatment

Control

3

Body wt (g +_ SEM ) 95 _+ 7

Alkaline phosphatase (umt/g +_ SEM)

Aryl sulfatase (umt/g + SEM)

4sCa Incorporated (cpm/mg _+ SEM)

58 0 6 +

156 32+_1183

5965_+845 6605_+762 7448+_520"** 581 _+152"*

288

PVP (10 mg/kg) PVP (100 mg/kg) EHDP(10 mg/kg)

105 +_ 5 98 + 8 107 _+4

67 87_+ 5 4 0 64 05+_ 3 27 82 65 +_ 16 50

179 23_+26 29 198 71_+21 10" 73 36 _+ 16 63*

PVP (100 mg/kg) EHDP(10 mg/kg)

121 + 5 114 _+ 5

67 70_+ 6 8 3 80 85_+ 8 8 3

212 6 7 + 3 0 0 7 7503+_752 55 9 6 + 1 1 60** 2568+_648.

pronounced at 7 days of treatment than at 4 days (Table II). A slgmflcant decrease m aryl sulfatase, but not of alkahne phosphatase, was also evident following EHDP treatment (Table II). EHDP treatment for 7 days slgmficantly reduced both calcmm and phosphate content of the ossicle, the p h o s p h a t e c o n t e n t b e i n g a f f e c t e d t o a g r e a t e r e x t e n t { T a b l e I). O n t h e other hand, PVP either at a dose of 10 or 100 mg/kg had no effect on any parameter of bone calcification at any time. On the contrary, a somewhat higher activity of aryl sulfatase seemed evident at the 100 mg/kg dose (Table II). T h e m v i t r o s t u d y o f t h e e f f e c t o f E H D P o r P V P o n 4SCa i n c o r p o r a t i o n b y t h e o s s m l e s p r o d u c e d i n t e r e s t i n g r e s u l t s ( T a b l e I I I ) . W h i l e E H D P signific a n t l y b l o c k e d t h e i n c o r p o r a t i o n o f 4SCa i n t o m i n e r a l , i t h a d n o e f f e c t o n t h e e x c h a n g e a b l e c a l c m m , as c o m p a r e d t o c o n t r o l s . O n t h e o t h e r h a n d , P V P TABLE III IN VITRO E F F E C T OF PVP OR EHDP ON THE INCORPORATION OF "SCALCIUI~ BY THE OSSICLES The results represent mean of 4 separate experiments ,n each group *Not slgmfmant, **P < 0 01 Group

Treatment

Exchangeable 4sCa (cpm/mg (× 10 '") -+ S E M )

4sCa Incorporated ( c p m / m g ( × 1 0 k ' ) +_S E M )

1 2 3

Control EHDP (1 mg/ml) PVP (1 mg/ml)

10 40 _+ 1 02 8 14 +_ 1 17" 14 50 +_0 12"*

7 77 +_0 78 4 75 _+0 32** 6 65 _+0 56*

344 TABLE IV IN VITRO EFFECT OF PVP AND EHDP ON ALKALINE PHOSPHATASE AND ARYL SULFATE Enzyme

Additive

Activitya

Relative actlwty

Alkaline phosphatase

None

0 032

100%

PVP(1 mg/ml) PVP (10 mg/ml) EHDP (1 mg/ml PVP (10 mg/ml) ~CaC12 (10 mM) EHDP (1 mg/ml) + CaC12 (10 mM) None PVP (1 mg/ml) PVP (10 mg/ml) EHDP (1 mg/ml) PVP (10 mg/ml) + CaCl2 (10 mM) EHDP (1 mg/ml) + CaC12 (10 mM)

0 0 0 0

Aryl sulfatase

017 010 018 025

52% 31% 56% 78%

0 023

72%

0 0 0 0 0

030 017 007 019 021

100% 57% 23% 63% 70%

0 025

83%

aEnzyme actlvltms are denoted as mmromoles of p-mtrophenol or p-mtrocatechol liberated at 37°C m 30 mm by 0 1 ml of tissue extract from day 14 control ossmle using duplicate determinations caused a slgnifmant r e t e n t i o n o f e x c h a n g e a b l e 4SCa b y the ossmles even d u r i n g this s h o r t t r e a t m e n t , while it did n o t alter 4SCa fLxatlon into minerals (Table III) In vitro a d d i t i o n o f E H D P o r PVP t o e n z y m e a h q u o t s significantly inh i b i t e d the actlvltms o f b o t h aryl sulfatase and alkaline p h o s p h a t e w h m h were partially r e s t o r e d b y a d d i t i o n o f e x o g e n o u s calcium (Table IV). Hlstologmal e x a m i n a t i o n o f ossmles t r e a t e d with either E H D P or PVP for 7 days m d m a t e d n o r m a l osteogenesis b y the presence o f osteoblasts and o c c a s i o n a l l y islands o f c h o n d r o l y t m focl. However, a r e m a r k a b l e widening o f tibml eplphyses was visually d l s c e r m b l e and n o t e d at 7 days of t r e a t m e n t with E H D P , an e f f e c t n o t f o u n d f o l l o w i n g PVP t r e a t m e n t . DISCUSSION O n t h e basis o f earher o b s e r v a t i o n s w h i c h indicated t h a t PVP shares certain c o m m o n p r o p e r t m s with d l p h o s p h o n a t e s , such as high a d s o r p t i o n a f f l m t y to m m e r a h z e d m a t r i x surface [ 1 0 , 1 1 ] resistance to d e g r a d a t i o n by e n d o g e n o u s p h o s p h a t a s e and their ability to m h l b l t t r a n s p o r t o f Ca :+, b u t n o t t h a t o f p h o s p h a t e or fluoride, it was o f interest t o investigate the effect o f PVP o n calcification m vlvo. It was f u r t h e r assumed that if the p r i m a r y m o d e o f a c t i o n o f d l p h o s p h o n a t e s o n calcification is d u e to their physlcoc h e m m a l p r o p e r t m s t h a t p r e v e n t h y d r o x y a p a t l t e crystal g r o w t h , the poly-

345 menc phosphonates may accomphsh the same action as dlphosphonates [6,7]. Our results mdmate that, at a comparable or at a much higher dose, PVP failed to influence any parameter of calcification of ectopm bone. On the other hand, EHDP invariably interfered with calcification as determined by 4SCa-mcorporatlon into minerals, the effect being more pronounced dependmg on the duration of treatment. These findmgs confirm the observations of other mvestlgators who found an inhibitory effect of dlphosphonates on bone calclfmatlon [3--5,22,23] including a study of Plasmans et al. [24], who concluded that osteo-mductlve capacity of demmerahzed bone implant was not affected by EHDP while calcffmatxon and remodehng were impaired. In our study we observed a suppressive effect of EHDP on aryl sulfatase, but not on alkaline phosphatase activity. Our own observations, as well as studms of other laboratories, have shown that aryl sulfatase may be linked to degradation of the proteoglycan matrix, which occurs during bone remodeling and mineralization [18,25,26]. Thus, an inhibition of sulfatase by EHDP m vlvo is m agreement with observations of other investigators who observed that these compounds affect bone remodehng [22]. However, PVP which did not bring about any affect on sulfatase at the same dose, also fmled to affect calcification. At a higher dose (100 mg/kg) PVP appeared to increase sulfatase actlwtms which could be due to a higher number of macrophages infiltrating the implant under t h a t condition. These data suggest that an lntracellular mode of action may be required rather than a physmochemmal effect of polyphosphonates on h y d r o x y a p a t i t e to exert an effect on mineralization. The extracellular physicochemmal effect may still predominate the desorption of the mineral. It has been shown that EHDP also interferes with certain biochemical processes involving carbohydrate metabolism [9,10] as well as calcium transport [27,28]. Though it is presumed t h a t calcium sequestration m a y lead to interference with some metabolic processes [8], this seems not to be the main mechamsm accounting for the inhibition of aryl sulfatase in wvo, because a much higher dose of PVP fails to bring about any effect on the activity of this enzyme. On the other hand both PVP and EHDP inhibited alkaline phosphatase and aryl sulfatase in vitro. It seems, therefore, that an in VlVO effect of dlphoshonates on calcification as well as bone resorption is mediated through lntracellular mechanisms of action. Because of its high polyamonic and polymeric property, PVP is not likely to have access into cells and may be unable to influence the internal metabolic processes which eventually affect calcification or resorption of bone, although it is strongly adsorbed to mmerahzed matrLx. The higher affinity of PVP to mmerahzed surface is indirectly supported by the observation that under identical condltions, the PVP-treated ossmles bound slgmfmantly more 4SCa while having no effect on its incorporation into minerals. On the other hand, EHDP treated ossmles did not bind more 4SCa, but still affected its mcorporatmn into minerals even d u n n g this short exposure. In conclusion, our studies suggest that in addition to an external mechanism of actmn rendered by thetr physlcochemmal properties an lntracellular

346 m e c h a n i s m m a y c o n t r i b u t e t o t h e e f f e c t o f d l p h o s p h o n a t e s o n b o n e calcif i c a t i o n a n d r e s o r p t i o n . P V P , w h o s e a c t i o n is p o s s i b l y r e s t r m t e d t o t h e e x t r a c e l l u l a r c o m p a r t m e n t , IS u n a b l e t o a f f e c t b o n e m i n e r a l i z a t i o n u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . H o w e v e r , i t is still c o n c e i v a b l e t h a t p r o l o n g e d t r e a t m e n t w i t h p o l y m e r i c p o l y p h o s p h o n a t e s will a f f e c t b o n e m e t a b o h s m b y a f f e c t i n g c a l c i u m b a l a n c e a n d b y m a k i n g t h e e x t r a c e l l u l a r m i n e r a l less p r o n e to dissolutmn. ACKNOWLEDGEMENT T h i s s t u d y w a s s u p p o r t e d b y a g r a n t f r o m N I H 5 R 0 1 D E 0 5 3 2 1 t o M. Anbar. REFERENCES

1 R G G. Russel and H Flelsch, Pyrophosphate and dlphosphonates in skeletal metabohsm, Clm O r t h o , 108 (1975) 241 2 M D Francis and R L Centner, The development of dlphosphonates as slgmhcant health care products, J Chem E d u c , 55 (1978) 760 3 R G G Russel, R Smith, C Preston, R J Walton and G C Woods, Dlsphosphonates m Paget's disease, Lancet (1974) 926 4 O L M B]jvoet, A J G Nollen, T J J H Sloof and R Felth, Effect of a dlphosphonate on para-artmular osslfmatlon after total hip replacement, Acta Ortho Scand, 45 (1974) 926 5 R E Canheld, T P Jacobs and E S Sins, Chnmal apphcatlons of dlphosphonates, m Cohn, Talmage and Mathews (Eds), Hormonal Control of Calcium Metabohsm, Excerpta Medica, Amsterdam, 1981, p 124 6 R C Muhlbauer and H. Flelsch, Effects of dlsphosphonates on glycosammoglycan synthesis and proteoglycan aggregation m normal adult articular eartdage, Arthritis, R h e u m , 21 (1978) 97 7 M Palmoskl and K Brandt, Effects of dlphosphonates on glycosaminoglycan synthesis and proteoglycan aggregation m normal adult articular cartilage, Arthritis R h e u m , 21 (1978) 97 8 D K Fast, R Fehx, C Dowse, W F Newman and H Flelsch, The effects of dlphosphonates on growth and glycolysls of connective tissue cells m culture, Blochem J , 172 (1978) 97 9 R Felix and H Flelsch, Incease m fatty acid oxidation m calvaslal cells cultured with dlphosphonates, Bmchem J , 196 (1981) 237 10 M. Anbar, G A St John and A C Scott, Orgamc polymeric polyphosphonates as potential preventive agents of dental caries In vitro experiments, J Dent Res, 53 (1974) 867 11 M Anbar and G A. St John, Adsorption of polyphosphonated polyethylene m enamel o f t e e t h , J Dent R e s , 3 ( 1 9 7 1 ) 7 7 8 12 M Anbar, E P Farley, D D Denson and K R Maloney, Localized fluoride lelease from fluorine carrying polyphosphonates, J Dent Res, 58 (1979) 1134 13 A H Reddl and C B Huggms, Biochemical sequences m the transformation of normal flbroblasts m adolescent rats, Proc Natl Acad Scl U S A , 69 (1972) 601 14 A H Reddl and W A Anderson, Collagenous bone matrix-reduced endochondral ossffmatmn and hemopomsls, J Cell Blol , 69 (1976) 557 15 N C Rath and A H ReddL Influence of adrenalectomy and dexamethasone on matllx reduced endochondral bone differentiation, Endocrinology, 104 (1979) 1698

347 16 N C Rath and A H Reddl, Evaluation of anti-inflammatory drugs based upon the inhibition of matrix-reduced ornithme decarboxylase activity during connective tissue proliferation, Proc Soc Exp Blol Med., 162 (1979) 320 17 P.S Chen, J r , T Y. Torlbara and H Warner, Mmrodeterminatlon of phosphorus, Anal Chem, 28 (1956) 1756 18 N C Rath, A R Hand and A H Reddl, Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage, bone and bone marrow development, Dev Blol, 85 (1981) 89 19 N C Rath, D Meckler and M Anbar, Effect of dietary calcium deficiency on ectoplcally induced bone, Nutr Res, 2 (1982) 481 20 H C Anderson and S W Sajdera, Calcifmatlon of rachltic cartilage to study matrix vesicle function Fed Proc, 35 (1975) 148 21 N C Rath and M Anbar, Effects of adrenalectomy and aldosterone on mmerahzatlon of ectopmally-mduced bone, m M Silberman and H C Slavkm (Eds), Current Advances in Skeletogenesls, Excerpta Medici, Amsterdam, 1982, p 136 22 S C Miller and W S S Jee, The comparative effects of dlchloromethylene dlphosphonate (CIMDP) and ethane-l-hydroxy 1,1-dlphosphonate ( E H D P ) o n growth and remodelhng of rat tibia, Calclf Tissue Res, 23 (1977) 207 23 H Flelsch and R Felix, Dlphosphonates, Calclf Tissue I n t , 27 (1979) 91 24 C M T Plasmans, W Kuypers and T J J H. Sloof, The effect of ethane 1-hydroxy1,1-dlphosphonic acid (EHDP) on matrix induced ectopic bone formation Clln Orthop Relat Res, 132 (1978) 233 25 J Thyberg and U Frmberg, Lysosomal system m endochondral growth, Prog Histochem. Cytochem., 10 (1978) 301 26 D Bayhnk, J Wergedal and E Thompson, Loss of protein polysaccharldes at sites where bone mmerahzatlon is lmtmted, J Hlstochem Cytochem, 20 (1972) 279 27 R A Evans, D.J Bayhnk and J Wergedal, The effects of two dlphosphonates on bone metabohsm m rat, Metab Bone Dis Rel Res, 2 (1979) 39 28 D F Gufiland, J D Sallls and H Flelsch, The effect of two dlphosphonates on the handhng of calcium by rat kidney mitochondrm m wtro, Calcif Tissue Res, 15 (1974) 303