Calc. Tiss. Res.

3, 151--162 (1969)

The Inhibition of Calcium Hydroxyapatite Crystal Growth

by Polyphosphonates and Polyphosphates
MAI~ION D. FRANCIS
Miami Valley Laboratories, The Procter & Gamble Company, Cincinnati, Ohio

Received August 3, 1968

The formation of crystalline calcium hydroxyapatite from solutions of eMcium and phos-
phate ions and the inhibition of eMcium hydroxyapagite crystal growth by polyphosphonates
and polyphosphates have been studied. The polyphosphonates, disodium ethane-l-hydroxy-
1,1-diphosphonate and disodium diehloromethane diphosphonate, are effective inhibitors of
calcium hydroxyapatite erystM growth. The polyphosphates are also effective inhibitors of
calcium hydroxyapatite crystal growth as long as the required level of intact polyphosphate
is present in the system. However, because of their hydrolytic instability, which is enhanced
by high temperature, low pH, and certain enzymes, the concentration of the polyphosphate
decreases with time in vitro, and its activity as an inhibitor is lost. In contrast to the poly-
phosphates, the polyphosphonates are hydrolytieMly stable. The polyphosphonates are
ehemisorbed on the surface of the mierocrystMlites of calcium hydroxyapatite and, in the
manner of other known crystal growth poisons, thus prevent further crystal growth. The
stability of the polyphosphonates and their chemisorption on apatite suggest their use in
medical and dental applications involving pathological calcium and phosphate metabolism.
Key words: Calcification - - Physiologic - - Phosphonie Acids - - Phosphates - - Crystalliza-
tion - - Electron Microscopy.

On a 6gudi6 la formation de l'hydroxyapatite de calcium eristallin s partir de solutions

d'ions de calcium et de phosphate et l'inhibition de la eroissanee de cristaux de l'hydroxy-
apatite de calcium au moyen de polyphosphonates et de polyphosphates. Les polyphospho-
nages, 6thane hydroxy-l-diphosphonate-l,1 de disodium et diehlorom6thanediphosphonate de
disodium, song inhibiteurs effieaees contre la croissance de eristaux de l'hydroxyapatite de
calcium. Les polyphosphates sont aussi inhibiteurs efficaces contre la eroissanee de eristaux
de l'hydroxyapatite de calcium tang que le niveau exig6 de polyphosphate intact est pr6sent
duns le systgme. Cependant, s cause de leur instabilit6 hydrolytique, qui est soulign6e par
une temp6rature 61ev6e, valeur de pH basse, et eertaines enzymes, la concentration du poly-
phosphate diminue avec le temps in vitro, et son aetivit6 eomme inhibiteur est perdue. Au
eontraire aux polyphosphates, les polyphosphonates song hydrolytiquement stables. Les
polyphosphonates song chimiosorb6s sur la surface des microeristallites de l'hydroxyapatite
de eMeium, ainsi emp~chant l'occurrence d'autre croissanee de eristaux semblable s Faction
d'autres poisons eonnus de croissanee de eristaux. On propose l'extension de eette action sur
la formation de l'apatite et cette stabilit6 des polyphosphonates aux applications m6dicMes
et dentaires eoncernant le metabolisme pathologique de calcium et de phosphate.

Die Bildung des kristMlinen Calciumhydroxyapatit aus LSsungen, welche Calcium- und
Phosphationen enthalten, und die Hemmung der Bildung yon kristMlinen Caleiumhydroxy-
apatit dutch Polyphosphonate und Polyphosphate wurden ungersueht. Polyphosphonate,

]?or reprints: Dr. MA3alO~r D. F~A?CClS,Miami Valley Laboratories. The Procter & Gamble
Company, P.O. Box 39175, Cincinnati, Ohio 45239.
152 M.D. FRANCIS:

Dinatriumiithan-l-hydroxyl-l,l-diphosphonat und Dinatriumdichloromethandiphosphonate

verhindern das Kristallwaehstum des Calciumhydroxyapatits. Die Polyphosphate verhindern
ebenfa.lls das Kristallwaehstum des Caleiumhydroxy~patits, solange die notwendige Kon-
zentration des nicht hydrolysierten Polyphosphats vorhanden ist. Wegen ihrer hydroIytischen
UnbestEndigkeit, die durch hohe Temperatur, niedrige pit und bes~immte Enzyme erh6ht
wird, vermindert sich jedoch die Konzentration des Polyphosphats ~llm/~hlich in vitro, und
ihre Hemmungsaktivit~t geht verloren. Im Gegensatz zu den Polyphosphaten sind die Poly-
phosphonate hydrolytisch best~Lndig. Die Polyphosphonate werden an der Oberfl~Lche der
Mikrokristallite des Calciumhydroxyapatits ehemisorbiert und verhindern, wie andere be-
kannte Kristallwachstumsgifte, auf diese Weise weiteres Kristallwachstum. Die BestEndigkeit
der Polyphosphonate und ihre Chemisorption an dem Apatit empfehlen ihren Gebrauch in
der grztlichen und zahnErztlichen Praxis, soweit sie den p~thologischen Calcium- und Phos-
phatstoffwechsel betreffen.

Introduction
The formation of calcium phosphate solids is of fundamental importance to
man because calcium phosphate in the form of calcium hydroxyapatite (HA) is
the main constituent of the skeletal system. Furthermore, the deposition of cal-
cium phosphate, again primarily as HA, in regions of the body which should not
calcify, is encountered in m a n y pathological conditions such as dental calculus,
bursitis, arthritis, and m a n y other forms of cctopic calcification (SELYE, 1962).
Numerous workers (BgowN, 1966; MAoGREGOI~and BROWN, 1965; EA~ES et al.,
1965; GLIMCI~E1L1965 ; and WALTON et al., 1967) have investigated the mechanism
of formation of H A or bone mineral under solution conditions, but there is no
complete agreement on the route by which calcium and orthophosphate ulti-
mately become the crystalline material called hydroxyapatite. There is general
agreement, however, on the fact t h a t when calcium and orthophosphate are
mixed together at high p H the solid formed is initially amorphous, as defined b y
a lack of ability to obtain diffraction patterns by the physical methods of X - r a y
and electron diffraction. The latter technique suggests the lack of organized
atomic layers of greater than about 2 to 3 unit cells (BIENENSTOCKand POSNEg,
1968). The role of pyrophosphate, tripolyphosphate, and long chain phosphates
in inhibiting the formation of H A in vitro and in vivo has been clearly shown by
FI~EISC~I and co-workers (FI,ElSClt and NEUMAN, 1961; Fn]~IscHetal., 1965;
II~VI~G etal., 1966; SOHIBLE~ and FLEISCI~, 1966; and others, N]~WESELY, 1967;
TUOHWS~BERand GABBIA~I, 1967). The purpose of this paper is to show that poly-
phosphates and polyphosphonatcs inhibit specifically the crystal growth of H A
and to show that the effects of these two materials on H A crystal growth differ
by virtue of differences in their hydrolytic stability. This fundamental difference.
between the polyphosphates which hydrolyze and lose their ability to inhibit
crystal growth of H A and polyphosphonates which do not, suggests the medical
and dental application of the polyphosphonates to the treatment of pathological
calcium and phosphate metabolism.

Methods
A stock solution of calcium chloride was made from CaCI~-2H20; the orthophosphate
solution was made from NaH2PO~. H20. Equal volumes of the solutions were mixed together
with agitation under nitrogen and were kept at constant pH, 7.4 or 12.0 by means of a l~adio-
meter pH star apparatus (Radiometer, 72 Emdrupraj, Copenhagen NV, Denmark; The Lon--
 Inhibition of Calcium Hydroxyapatite Crystal Growth 153

don Co., Westlake, Ohio) until time for recovery of the precipitate. The solutions containing
precipitated calcium phosphate were filtered through 0.45 ~ Millipore filters and the precipi-
tates were air dried and analyzed or were embedded in methylmethaerylate, sectioned, and
examined by electron microscopy and electron diffraction in a Siemens Elmiskop I (Siemens
und Halske, Aktiengesellschaft, Wernerwerk fiir MeBtechnik, Karlsruhe, Germany; Siemens
American, New York City). When materials were added to inhibit the crystal growth of
hydroxylapatite, they were usually added to the phosphate solution, then the calcium solu-
tion was added to produce instantaneous precipitation, except where indicated otherwise.
Concentrations of solutions and reaction conditions are listed in the legends to the figures.
Calcium analysis was performed with a Perkin-Elmer, Model 303 atomic absorption spectro-
meter (Perkin-Elmer, Norwalk, Conn.). Phosphate was determined by various combinations
of three methods depending upon whether only orthophosphate (MARTInand DoTY, 1949),
combined ortho- and polyphosphate (LucE~A-CosDEand PRAT, 1957), or total phosphorus
(ortho- plus phosphonate) was desired. In the latter case, the samples were mixed with KHSO~
and fused until the melt solidified; the latter was then taken into solution with acid, as usual,
and analyzed for combined ortho- and polyphosphate (LucENA-Co~DEand PRAT, 1957).
The inhibitors of crystal growth used were tetrasodium pyrophosphate deeahydrate,
pentasodium tripolyphosphate, disodium ethane-l-hydroxy-l,l-diphosphonate (EDHP), and
disodium dichloromethanediphosphonate (C12MDP). EHDP-I-~4C was synthesized from
acetate-l-~4C by the method of QuIMBYand P~ENTICE (1966) and was used in adsorption
studies.

Results
When calcium and orthophosphate solutions are mixed together and held at
constant p H of 7.4 for 45 minutes at 25~ the precipitate formed is crystalline
H A as shown by the electron diffraction pattern (Fig. 1 a). The crystals formed are
needle-like as shown in the electron mierograph (Fig. 1 b). Some of the gel-like
spheres originally reported by WE]~ER et al. (1967) are still seen, however (Fig. 1 b).
If the same experiment is repeated but sodium pyrophosphate or sodium tripoly-
phosphate is added to the orthophosphate solution prior to addition of the calcium
chloride, no crystallites are formed and the entire mass of solid precipitate is
amorphous by electron diffraction (Fig. 2 a). This precipitate has the appearance
of aggregated clusters of small spheres (Fig. 2b) similar to the initial amorphous
form (W~B]~etal., 1967). I n another experiment the control calcium and
phosphate solutions at pH 7.4 were mixed and filtration was begun immediately.
The electron diffraction pattern (Fig. 3a) obtained from the precipitate was a
good crystal pattern of hydroxyapatite and the crystallite particles were readily
visible by electron microscopy (Fig. 3b). Crystals in this control system were not
as large as the crystals shown in Fig. l b because the precipitate was filtered im-
mediately rather than after 45 minutes of incubation. If E H D P is added to a
similar system (in the phosphate solution), crystal growth of HA is prevented, the
precipitate formed is amorphous by electron diffraction techniques (Fig. 3c),
and the precipitate has an agglomerated gel-like appearance (Fig. 3 d).
The important difference between the polyphosphonates and the polyphos-
phates with regard to their interference with the crystal growth of H A lies in the
hydrolytic stability of the polyphosphonates and the hydrolytic instability of the
polyphosphates. This difference is illustrated by the data shown in Fig. 4 (a, b, e,
d, e, f). I n this series of experiments the calcium phosphate precipitates were
formed at pH 7.4 in the presence of either pyrophosphate, tripolyphosphate,
E H D P , C12MDP, or no inhibitor (control). These were all incubated at 80 ~ for
154 M.D. F R ~ c m :

Fig. 1 a and b. The appearance of freshly precipitated calcium orthophosphate, a Electron

diffraction pattern of hydroxyapatite. The precipitate was formed by reaction of 1 • 10-2 M
CaC12, 1 • 10-2 M NaHePOt, pH -- 7.4, 45 minutes at 25~ b Electron micrograph of pre-
cipitate from (a) showing the long needle-like crystals of hydroxyapatite (• 190,000)

48 hours to facilitate hydrolysis. I n t h e control, no inhibitor, a good b u t s p o t t y

p a t t e r n for I-IA was o b t a i n e d b y electron diffraction (Fig. 4a). The large rectan-
gular crystals responsible for this p a t t e r n are seen in t h e micrograph, Fig. 4 b .
Fig. 4 e shows t h e diffraction p a t t e r n o b t a i n e d from t h e p r e c i p i t a t e f o r m e d in
t h e presence of p y r o p h o s p h a t e ; as in the control, a good b u t s p o t t y p a t t e r n for
H A was o b t a i n e d i n d i c a t i v e of the presence of large crystals as shown in t h e micro-
graph, Fig. 4d. Thus, i t is obvious t h a t while p y r o p h o s p h a t e is a n excellent in-
h i b i t o r of H A c r y s t a l growth, u n d e r t h e conditions of this e x p e r i m e n t it is de-
s t r o y e d b y hydro]ysis a n d H A crystals are t h e n free to form w i t h o u t interference.
W h e n t r i p o l y p b o s p h a t e was a d d e d to t h e s y s t e m t h e results were identical to
 Inhibition of Calcium Hydroxyapatite Crystal Growth 155

Fig. 2a and b. The appearance of freshly precipitated calcium orthophosphate inhibited by

tripolypbosphate ion. a Amorphous electron diffraction pattern. The precipitate was formed
by reaction of 1 • 2 ~ CaC12, 1 • - ~ NaH2PQ, l • -aN NasPaO10, pH7.4,
~5 minutes at 25~ b Electron mierograph of gel-like agglomerates of calcium phosphate
( • 190,000)

those with pyrophosphate shown in Fig. 4c, d. W h e n calcium phosphate was

precipitated in the presence of E H D P , however, there was no evidence of crystal
formation~ even after 48 hours incubation at 80 ~ The electron diffraction p a t t e r n
for this precipitate shows it to be amorphous (Fig. de) indicating no organized
atomic (or ionic) arrangements within the resolving power of this technique. The
electron micrograph (Fig. 4f) also shows no indication of crystallinity, thus E H D P
was totally effective in inhibiting the crystal growth of HA. W h e n CI~MDP was
added to the system the results were identical to those with E H D P as shown in
Fig. 4 e, f.
I n order to investigate the nature of the inhibition of H A crystallization, two
systems involving a "crossover" of filtrate and precipitate were set up as shown
11 Calm Tis~. Res., Vol. 3
156 M.D. FRAI~CIS:

s 3 a--d
 Inhibition of Calcium Hydroxyapatite Crystal Growth 157

in Fig. 5. Calcium phosphate, Precipitate-l, from the control (with no E H D P ) and

similar crystallographically to precipitate in Fig. 3a, b was placed with Filtrate-2
(the filtrate from the calcium phosphate precipitated in the presence of E H D P ) .
Large crystals were obtained after 48 hour incubation (similar to those seen in
Fig. 4b), and a s p o t t y H A p a t t e r n was obtained indicating t h a t the Filtrate-2
did not contain sufficient E H D P to affect crystal growth (less t h a n 1 X 10 -5 M).
I n the other possible combination, Precipitate-2 (the calcium phosphate precipi-
tate in the presence of E t t D P ) and similar to precipitate in Fig. 3 e. d, was com-
bined with Filtrate-1 from the control. I n this case a stable colloid was formed in
8 to 16 hours and after 58 hours crystal growth of H A was still almost completely
inhibited. I n this experiment crystal]ires of H A from Precipitate-2 incubated in
Filtrate-1 did grow sufficiently to give a H A electron diffraction pattern. Electron
microscopy, however, failed to show a n y distinct crystallites (the particles were
similar to those shown in Fig. 4f). Thus, E H D P was principally associated,
coprecipitated, or occluded with Precipitate-2, not with Filtrate-2.
To understand further the association of E H D P with the preeipitafe of
calcium phosphate, the distribution of EHDP-l-14C between precipitate and
filtrate was investigated. The results are shown in Table 1. W h e n calcium and
orthophosphate concentrations, temperature, time, and p H were held constant
the 14C-EHDP was always predominately associated with the precipitate (88.0 to
97.6%). This was true even when the concentration of the E H D P was 5 x 10 s M
( < 0.02 p p m E H D P ) .

Table 1. Distributiort o/ EHDP-1-14C between precipitate and filtrate (pH 7.40, 60 minutes
reaction, [CaC12] ~ 4.0 • 10 -3, [NaHeP04] = 4.0 • 10 -3)

Concentration precipitate Filtrate

of EHDP-I-~4C
(m/l) cpm % of total cpm % of total
recovery recovery

1.2 • 10-4 246,000 97.6 6,000 2.4

3.0 • 10-5 74,500 95.1 3,800 4.9
1.0 X 10-6 56,000 94.0 3,700 6.0
5.0 X 10-s 4,520 88.0 600 12.0

The effect of E H D P and Cl~MDP on the rate of precipitation of calcium phos-

phate is shown in Table 2. W i t h similar conditions and times of precipitation, the
concentration of calcium in the filtrate was about 33 and 8.4 times higher and
phosphorus about 1.7 and 2.0 times higher in systems containing the two effective
crystal growth inhibitors, E H D P and C12MDP, respectively, t h a n in the controls.

Fig. 3a--d. Structure modifications of calcium orthophosphate precipitates, a. Electron dif-

fraction pattern of hydroxyapatite. The precipitate was formed from 1 X 10-2 M CaC12,
1 X 10-2 M NaH2PO4, pH 7.4 at 25~ b. Electron micrograph of precipitate (a) showing very
small needle-like crystals just forming from original amorphous precipitate, c. Amorphous
electron diffraction pattern, d. Electron micrograph of precipitate (c) showing gel-like
agglomerates, b and d x 150,000
11"
156 M.D. FRAI~CIS:

s 3 a--d
 M. D. F~A~elS: Inhibition of Calcium Hydroxyapatite Crystal Growth 159

Fig. 4 e and f
Fig. 4a--f. The effect of two different crystal modifiers on calcium orthophosphate precipita-
tion. a Spotty electron diffraction pattern of hydroxyapatitc. The precipitate was formed
from 1 • 10-2 M CaC12, 1 • l0 -~ M NaH2P04, pH 7.4, 48 hours reaction at 80~ b Electron
micrograph showing pronounced rectangular crystallites of hydroxyapatite, c Spotty electron
diffraction pattern of hydroxylapatite. The precipitate was formed from 1 • l0 2 M CaC12,
I • 10-2 M NaH2P04, 1 • l0 -a M Na~P20~, pH 7.4, 48 hours at 80~ d Electron micrograph
showing pronounced rectangular crystallites of hydroxyapatite not different from (b).
e Amorphous diffraction pattern. The precipitate was formed from 1 • 10-e M CaCl~, 1 • 10-e M
Nail,P04, 1 • 10-aM EHDP, pH 7.4, 48 hours at 80~ f Electron micrograph of precipitate (e)
showing non-crystalline, gel-like nature of precipitate even after 48 hours of reaction time.
b, d and f • 180,000

The elevated calcium a n d phosphate concentrations p r o b a b l y result a t least i n

p a r t from the extremely small crysta]lite size of H A (amorphous) in the presence
of the inhibitors as borne out b y the electron mierographs a n d diffraction p a t t e r n s
(Figs. 2, 3c, d, a n d 4c, f). These data illustrate the p r e v e n t i o n of the removal of
calcium a n d phosphate from the solution via precipitation b y the inhibitors of
crystal growth as a result of rate considerations or crystal size affecting solubility
factors.
160 M. D. FRA~CZS:

Ca--P Precipitate Sorption of EHDP

~ Precipitate-- I I A Precipitate -2 I
IOa+P+EHDPI
"1 Fi,tr~ , I XNi Filtrate-2 I

I Precipita~e- I NO COLLOID
Filtrate -- 2 LARGECRYSTALS

I Precipitate -- 2 STABLECOLLOID
Filtrate -- I SMALL CRYSTALS

Fig. 5. A filtrate-precipitate crossover experiment. Both precipitations were carried o(]t at

1 • 10-2M CaC12, 1 • 10-2M NaH2PO 4, pH 12.0, but system No. 2 had added 1 • 10-aM
EHDP. The reagents were added together and the systems were filtered immediately. Fil-
trates and precipitates were then combined as shown. The resultant mixtures were then
incubated at 80~ for 48 hours and then centrifuged. Only the Precipitate-2-Filtrate-1 system
showed inhibition of crystal growth

Table 2. Terminal Ca und P concentrations as a/unction o] the inhibitor o/crystal growth

Inhibitor Inhibitor Initial Ca and P (m/l) Terminal Ca and P (m/l) Reaction

concen- time
tration Ca P Ca P (minutes)
(m/l) ( • 10a) ( X 10a) ( X 108) ( X 10a)
( • 10a)

EHDP 0.200 3.87 4.00 1.55 2.81 1,080

None -- 4.20 4.00 0.0468 1.69 1,176
C12MDP 0.200 4.20 4.00 1.60 2.56 164
None -- 4.20 4.00 0.191 1.31 120

Discussion
FLEISCK eta[. (1966) have shown t h a t p y r o p h o s p h a t e a n d polyphosphates are
readily hydrolyzed i n the presence of phosphatase or when a d m i n i s t e r e d in vivo
b y the oral route (FLEISCH eta[., 1966; FL]~zsc~eta[., 1968). I t is this r e a d y
hydrolysis of the polyphosphates which prevents the use of p y r o p h o s p h a t e a n d
other polyphosphates in the t r e a t m e n t of anomalous calcification problems in
m a n . As shown in the present experiments, however, the polyphosphonates EI-IDP
a n d CI2MDP do n o t hydrolyze a n d so r e t a i n their a b i l i t y to i n h i b i t H A crystal
growth u n d e r conditions in which polyphosphates would be t o t a l l y destroyed.
The effective control of heterotopie calcification in tissues such as aorta b y injec-
t i o n a n d b y oral a d m i n i s t r a t i o n of polyphosphonates to rats has been demon-
s t r a t e d b y FgA~CIS et al. (in preparation).
 Inhibition of Calcium Hydroxyapatite Crystal Growth 161

The crossover e x p e r i m e n t (Fig. 5) m i g h t suggest t h a t E H D P p r e v e n t s crystal

growth of H A b y physically covering the calcium orthophosphate precipitate b y
the f o r m a t i o n of a calcium E H D P which t h e n coprecipitates with the calcium
orthophosphate a n d p r e v e n t s crystal growth of the HA. F r o m Table 1, however,
it is e v i d e n t t h a t at E H D P concentrations far below those at which the calcium
E H D P can precipitate (10 -6 a n d 10 s M E H D P ) the EHDP-I-~aC was still entirely
associated with the calcium orthophosphate precipitate. I t is suggested, therefore,
t h a t the m e c h a n i s m of the i n h i b i t i o n of crystal growth is one of strong chemisorp-
tion of E H D P on the mierocrystallites of H A (possibly a t screw dislocation sites)
thus preventing the rapid addition of ions into the apatite lattice as occurs in the
absence of the inhibitor. As judged by electron diffraction, addition of ions into
the HA lattice in the presence of EHDP must not go beyond a few unit cells or
the organized structures would have been detected. This limiting of the crystallite
size probably also accounts for the colloidal suspension observed in the crossover
experiment. The ehemisorption properties of the EHDP also suggest the possible
use of these polyphosphorates for therapy of osteoporotic problems in man and
animals.

The author is grateful to 1~. L. STEWAI~Tfor the synthesis of disodium-l-hydroxy-l,l-di-

phosphonate-l-14C and to 1~. J. NEAL for the electron micrographs and diffraction patterns,
and to L. FLOI%Aand R. HICKS for technical assistance.

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