Selective inhibition of crystal growth on octacalcium phosphate and nonstoichiometric hydroxyapatite by pyrophosphate at physiological concentration

Journal of Crystal Growth 113 (1991) 643—652 North-Holland

643

Selective inhibition of crystal growth on octacalcium phosphate and nonstoichiometric hydroxyapatite by pyrophosphate at physiological concentration N. Eidelman, W.E. Brown American Dental Association Health Foundation, Paffenbarger Research Center, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA

and J.L. Meyer Nd, National Institutes of Health, Bethesda, Maryland 20892, USA

Received 22 February 1991

Octacalcium phosphate, Ca

5H2(P04)65H20 (OCP), appears to be a precursor in biomineral formation. The formation of OCP as the precursor is supported by the observation that stoichiometric hydroxyapatite, Ca5(P04)30H (OHAp), cannot form directly because of the presence of its growth inhibitors in serum. Therefore, the effects of the physiological concentration of pyrophosphate (P2O~),one of the most important calcium phosphate growth inhibitors in blood, on calcium phosphate growth rates on OCP and nonstoichiometric OHAp (apatite) seeds were measured. The amounts of seed crystals used to initiate the growth were adjusted by trial and error so that the control growth rates (in the absence of P2O~)were the same on both OCP and apatite seeds at a given supersaturation. The crystal growth on both kinds of seed crystals from supersaturated solutions in the presence of l1sM P2O~ added once (“one-time” addition) at constant pH (7.4) and 25°C was determined by KOH titration and decreases in Ca and P04 concentrations in the solutions. Crystal growth on OCP seed crystals in the presence of a constant concentration of 1~sMP2O~ was also measured. The growing phases were characterized by ~Ca/~PO4 ratios, chemical potential plots, X-ray diffraction (XRD) and Fourier transform infrared (FTIR). The results of this study show that: (1) P2O~ ions inhibited the growth on the apatite seeds more than on the OCP seeds; (2) apparently OCP precipitated on both types of seeds, followed by its hydrolysis to a more apatite-like phase; (3) slower crystal growth was observed on OCP seeds in the presence of a constant physiological concentration of P2O~(19M) than in the “one-time” addition of P2O~.

1. Introduction Octacalcium phosphate, Ca8H2(P04)6 5H20 (OCP), has been suggested as a precursor that hydrolyzes later to a nonstoichiometric OHAp in biological systems: in bone [1—3],in dentin [3], in enamel formation [4—6],in cartilage calcification [7], in femurs of Wistar rats [8] and in ultrafiltered serum [9]. OCP has been detected in different cases of various pathological calcification: by elec0022-0248/91/$03.50 © 1991

—

tron diffraction in aortic calcification [10]; by infrared spectroscopy in human synovial fluid [11]; by XRD [12], electron diffraction [131 and SEM [14] in human dental calculus; by XRD, light microscopy and electron activation in calcinosis [15]; and by XRD, light microscopy and SEM in neuroepithelial suprastructures of the human inner ear [16]. OCP was also found in articular cartilage in patients on chronic renal hemodialysis [17] and in patients with periarthritis [18]. The

Elsevier Science Publishers B.V. All rights reserved

644

N. Eidelman el al.

/ Selective inhibition

of crystal growth on

OCP and OHAp

observed stability of OCP in pathological calcification may be due to the presence of Mg, citrate

scribed [25]. The Ca solution used for the crystal growth experiments contained 5mM Ca(N03)2,

and/or pyrophosphate in the biological fluids [19]. Stoichiometric hydroxyapatite, Ca5(P04)30H (OHAp), did not form de novo in ultrafiltered serum, apparently because of natural OHAp crystal growth inhibitors in blood [4,9]. Therefore, to understand the mineralization process fully, the effects of inhibitors on the relative growth rates of OCP and nonstoichiometric OHAp must be established. Pyrophosphate, P20~, is an important inhibitor of calcium phosphate growth in blood [20—23].It has been shown [20] that calcium phosphate did not grow on OHAp seeds in the presence of a constant physiological concentration of P20~. It has also been shown recently [24] that ,P20~ affected differently the growth of calcium phosphate on OCP and on well-crystallized (stoichiometric) OHAp seeds when the control growth rates (without P20~) were adjusted by weight or surface area of the seed crystals. Consequently, the objectives of this study were: (i) to compare the inhibition effects of a physiological concentration of P20~ on the growth of calcium phosphate on OCP and nonstoichiometric OHAp (apatite) seeds, (ii) to determine if OCP can grow in the presence of a steady physiological concentration of P20~, (iii) to determine the relative rates of crystal growth on OCP and apatite seed crystals, and (iv) to characterize the phase which grows on the surface of the seed crystals.

140mM NaCl and 4.2mM KCI. The P04 solution used for the crystal growth experiments contained 3.75mM KH2PO4, 140mM NaCI and 4.2mM KCI.

2.2. Preparation of seed crystals OCP seeds were prepared by hydrolysis of dicalcium phosphate dihydrate, CaHPO4 2H20 (DCPD), in distilled water that was added dropwise over a period of about 3 months at room temperature [26].The crystals had a Ca/P ratio of 1.33 ±0.02, and the X-ray powder diffraction pattern (XRD) and the Fourier transformed infrared (FTIR) spectra showed the characteristic lines of OCP (figs. 9A and bA). The apatite seeds were prepared by the slow, constant addition of stock calcium nitrate (Ca(N03)2) and potassium dihydrogen phosphate (KH2PO4) solutions to a vessel maintained at C by a circulating water bath and pH 7.40 maintained by a pH stat. In this manner, the apatite seed crystals to be used in subsequent experiments were prepared under the physiological conditions of ionic strength, pH and temperature. The seed crystals were allowed to age at least two weeks at pH 7.40 before use. The crystals had a Ca/P ratio of 1.54 ±0.01 and were shown by XRD and FTIR to be a defect OHAp similar to that formed in biological mineralization (figs. 9D and 1OD). 370

2. Materials and methods 2.3. Experimental procedure 2.1. Reagents and stock solutions

The reagents used in this study were analytical grade, and the solutions were prepared with deionized, distilled water. The titrant was 0.1N KOH solution. The P20~-containing solution was 100~tM 32P-labeled sodium pyrophosphate (Na4P207 Na 10H20). 4P207~10H20 was obtamed from New England Nuclear Corporation, Boston, MA. The anion exchange column and and the 32P0~ elution buffers for the resolution 3~P~0~ ions were prepared as of previously de.

Metastable supersaturated solutions were prepared by carefully mixing equal-weight amounts of the Ca and P04 solutions with rapid stirring in jacketed Pyrex cells using circulating water from a constant-temperature water bath at 25°C. The experiments were done at 25°Cin order to minimize the hydrolysis of the OCP seeds. The pH was adjusted to 7.4 by slow titration of KOH with a pH stat (Fisher, II, Titration Pittsburgh, PA). Titrimeter The calculated [27,28] Systems, negative logarithm of the ion activity product of OCP

N. Eidelman et a!.

/

Selective inhibition of crystal growth on

(pIAP0~~) was 44.7 and the pIAPOHAP was 46.9. Since these values are lower than the negative logarithm of the solubility products of these salts (pKSp0~~ 48.6 [29], pKspoHAP 58.6 [30], the final solutions were supersaturated with respect to OCP and OHAp. However, the supersaturation was low enough to prevent spontaneous precipitalion. Two kinds of experiments were performed: (1) P 20~ was added once to the solution before addition of seed crystals in the “one-time” addition The inhibitor was labeled with 32Pexperiments. isotope in selected experiments in order to the follow the P 20~ solution concentration. (2) Constant physiological P2O~ concentration (1~tM) was maintained in the solution after addition of OCP seed crystals. This was done by the following procedure: an initial amount of labeled P2O~ (1~tM)was added before the addition of the seeds. Then an equal amount was added 1 mm after the seeding and the start of the titration to correct for the instantaneous [20]. The 32P activity in theadsorption solution of wasP20~ measured immediately after the second P 2O~ addition, and based on that measurement, an amount of P20~ was added to adjust the level again to 1j.tM. This was repeatedwas until the level of labeled P2O~ in the solution nearly constant, The amounts of seed crystals used to initiate the growth were adjusted by trial and error so that the control growth rates (in the absence of P 2O~) were the same on both OCP and apatite seeds at a given supersaturation. The seed crystals were added after wasrecorded adjusted, and the titration volume andthe pHpH were simultaneously with time by a two-pen recorder. The rate of crystal growth was monitored by the amount of base (0.1N KOH) required to maintain a constant pH throughout the crystal growth process and by measuring periodically the Ca and Po 4 concentrations in the solution. Samples were withdrawn periodically from the solutions and centrifuged at 12,000 g in an Eppendorf centrifuge 5414 (Brinkman Instruments, Westbury, NY). An aliquot of the supernatant was then carefully removed from the centrifuge tube for Ca, P04 and P20~ analyses. Ca Ca—Arsenazo was determined cally as the III spectrophotometricomplex [31], and =

=

-

—

OCP

645

and OHAp

P04 was determined spectrophotometrically as the ammonium—phosphomolybdate blue complex [32]. Each Ca and P04 measurement represents the average of three independent analyses. lAPs for the various sparingly soluble calcium phosphates were calculated from the Ca and P04 concentrations and pH by use of an iterative procedure 2~) [27,28]. Chemical potential plots, were —log[(Ca (OH—)2] versus —log[(H~)3(PO~)], used to identify the growing solid phases L~Ca/L~P04 ratio is defined as:

L~Ca/LXPO4 [Ca0 =

—

Ca1]/[(P04)0

—

[33]. The

(P04)~I, (1)

where Ca0 and (P04)0 are the initial concentrations of Ca and P04 and Ca1 and (P04)1 are the concentrations of Ca and P04 at time t in the same solutions. This ratio is an indication of which calcium phosphate phase grown to the time when the sample washad taken. Theupstandard deviations of L~Ca/L~ P04 in the solutions were calculated by using a formula derived by the “propagation 32P of error formulas” method [34]. The measurements were placed imaliquots for mediately after centrifuging on the anion exchange columns and were separated to solutions containing 32P 32P0~ [25]. The con2O~ and centrations of the P20~ and PO~ resulting from P20~ hydrolysis in the solution were calculated from32P thecounts activityasofChernekov the i2~ isotope, measured radiation [35] bybya the scintillation counter (Beckman, LS9000, Fullerton, CA). The solid phase in the centrifuge tube was collected periodically, filtered through a cellulose membrane (0.22pm filter, Millipore, Bedford, MA), air dried, and subjected to XRD (automated Nuralko diffractometer, Netherlands, Cu Ka, X 1.5405 A) and FTIR (Bruker, model 113v, Hamburg, Germany) analyses. The final solid phases of selected radioactive experiments were retrieved, washed several times with distilled water, and dissolved in 0.1M HC1 [20]. Measurements of the radioactive levels of the resulting an 32P solutions gave 32PO~ estimate of the amount of 2O~ and present in the solid phase [20]. =

646

N. Eidelman ci a!.

/ Se!ective

inhibition of crystal growth on OCP and OHAp

3. Results 3.1. “One-time” addition of P,O~

3.0

j

0

OCP+IpM P

* *

ocp+1p~t P207 OCP+1/1M P207 OCP control OCP , control

207

(A) 0

The effects of P20~ on the growth of calcium phosphate were determined by monitoring the titrant volume of base required to maintain constant pH and the decrease of Ca and P04 concentrations with time after addition of seed crystals. The reproducibility of the seeded crystal growth experiments was established by replication under the same conditions and is shown in figs. 1 and 2. The two upper curves in fig. 1 are the titration curves of two controls (growth of calcium phosphate on OCP seeds without inhibitor). The three lower curves are the titration curves of experiments in which calcium phosphate grew on OCP seeds in the presence of b~sMP20~. The two lower sets of symbols in figs. 2A and 2B represent the decreases in Ca (fig. 2A) or P04 (fig. 2B) concentrations of the same two control experiments, while the three upper ones are from the three P20~-containingexperiments. Each set of symbols belongs to a different experiment, and each point is an average of three independent analyses of Ca or P04. ___________________—

—

2.0

.~

a

~

1 .0

0.0

(B)

OCP+lpM P207 ocP+ipz~tP20., ocP+1p~MP207 OCP , control OCP , control

0

* *

2.0

0

0

a ,~

0~

* 0

0

__________________________ 10 Time 100(miii) 1000 10000

Fig. 2.

Reproducibility of seeded crystal growth experiments measured by the decreases in (A) Ca and (B) P04 concentrationS with time: the two lower sets of symbols are for two control experiments (without P2O~), and the three upper sets are for experiments of growth on OCP seeds in the presence of

-- -

4—

0

1.5

0

~

1.0 0.5 0.0

1

OCP,corstrol 0CP+1~iM P207 0CP,cont,rol OCP+l1iM P207

~ -—

0

500

— ________--——

1000

1500

Time (mm) Fig. 1. Reproducibility of seeded crystal growth experiments measured by 0.IN KOH titration with time: the two upper curves are the titration curves for two control experiments (without P2O~),and the three lower curves are for experiments of growth on OCP seeds in the presence of 1~Mp2o~.

1~MP207

from with Curves OCP the solution of seed titration of the crystal inremoval the growth presence ofseen Ca experiment of b~sM P04 between P20~ titration P04 curves. are the curve shown titration These iscrystals in mirror a fig. results and 3. the Itimage show can removal be of direct the of and that relation Ca and the P04. The effects of P2O~ on calcium phosphate crystal growth on OCP and apatite seeds under similar experimental conditions are shown in fig. 4. The titration rate was slower with OCP seeds in the presence of P20~ than was the case for the controls, and the titration rate with apatite seeds was much slower than the titration rate with OCP seeds (fig. 4). As can be seen, the titration curves

N. Eide!man ci a!.

/ Selective inhibition

of crystal growth on OCP and OHAp

647

both types of seeds were also about the same. This

~2.5

suggests that the same phase was growing on both 0°

seeds. Since the crystal growth was measured by two independent methods that were in good agreement (pH titration and decrease in Ca and P0 4 0

*

Calcium

concentrations, see also fig. 3), it can be concluded that the calcium phosphate growth was inhibited

Phosphate

more on the apatite seeds than on the OCP seeds 0

0.0 0

10

100 Time (mm)

1000

10000

Fig. 3 Calcium phosphate crystal growth on OCP seeds in the presence of

l.OpM P2O~ measured

by 0.IN KOH titration

and decreases of Ca and P04 in the solution with time,

by the “one-time” addition of bfLM P20~”. The concentrations of P2O~ in the solutions and the simultaneous titration curves at the beginning of the crystallization process, as a function of time, are shown in fig. 5. As can be seen, the slope

of the KOH titration increased as P20~ disapof the controls and of the P2O~-containing experiments have almost the same final volume titrated after the induction time. Similarly, the decreases in Ca and P04 concentration (not shown) followed the same trend, similar to that described in fig. 2 (smaller in the experiments with P20~ and OCP seeds than for the controls and smaller in the P20~-containing experiments with apatite seeds than in the experiments with OCP seeds). The final concentrations of Ca and P04 in the control and P20~-containing experiments with

______

1.5

*

0

o

peared from the solution (fig. 5A) in the experiment with OCP seeds, while in the same time period the titration (growth) on apatite seeds was limited by the slow decrease of the P2O~ in the solution (fig. SB). The levels of the radioactive P2O~ in the solid phases of the experiments with

1.00

~..—

(A)

on

OCP

seeds

0.75

/

0.75 ---0---

[P207]

—*—

KOH titration

0.50-’

~‘0.50

OCP,control apatite,control OCP+ljjM

P207 0.25

1.0 0.00

~ ~tjte+1~MPsO7

0

(B)---0--on apatite [P207]

0.25 0.75

—*——

seeds

~

0.75 “O

KOH titration

0

0.50

0.50

0.25

0.25

0 0.0 0

1000

2000 3000 Time (mm)

4000

____________________________

Fig. 4. The effect of P20~— on crystal growth on 0CP and apatite seeds measured by 0.1N KOH titration (0.IM) with time: the two upper curves are the titration curves for two control experiments (without P20~’) of growth on OCP and on apatite seems, the middle curve is for growth on OCP seeds in the presence of 1pM P20~, and the lower curve is for growth on apatite seeds in the presence of 1pM P2O~.

0.00 0

I

I

tO 100 1000 Time (mm) Fig. 5. Titration curves of seeded crystal growth and levels of labeled P2O~ in the solutions as a function of time: (A) on OCP seeds; (B) on apatite seeds.

648

N. Eide!man ci of

/ Selective inhibition

OCP seeds were found to be high even after the crystals were washed and partial hydrolysis of the P 20~ took place at the surface of the OCP crystals. This is in conformity with results reported for OCP [24] and OHAp [20,21].

The average values of LXCa/zW04 and their standard deviations (error bars) of representative experiments with OCP and apatite seeds are shown

of crystal growth on OCP and OHAp

(A) on OCP seeds

C ‘—...

a

in fig. 6. The standard deviations of the initial values of the ~Ca/LXP04 are large because the ~Ca and L%P04 had a large relative error in the early stages of crystal growth (substraction of two large numbers). The standard deviations decreased with time, reflecting the decreased percent error as the difference between the initial and actual Ca

and P04 concentrations in the solution increased as the growth progressed with time. Values of

~Ca/z~P04 of controls and P20~-containingexperiments with OCP and apatite seeds are shown in fig. 7. The average values for the ~Ca/L~P04

ratios of the solid phases in the first day (fig. 7) were: 1.32 ±0.10 (n = 17) for the experiments with OCP seeds and 1.34 ±0.06 (n = 11) for the experi-

.

1.3,

1.1

•

o

0.9

1.4

,___}...............~.*_*

1~~Mp2o7

*

1,uM

P207

—

(B) on apatite seeds

1.5 0

*

‘

< 1.3 a

~...

* •

control 0 control A control ~ 1/2M P207

1.1 0.9

—

0

ments with apatite seeds. The values of the ~Ca/~1P04 increased slowly thereafter to 1.5. The chemical potential plots of the controls

control control

A

10

100

1000

10000

Time (mm) Fig. 7. The composition of the phase growing on OCP seeds (A) and on apatite seeds (B), as measured by the changes in ~1Ca/dP04 with time in the solutions: controls and 1pM P2O~-containingexperiments. Each set of symbols belongs to a different experiment, and each point is an averaged value of and P04. z~Ca/zlP04calculated from three independent analyses of Ca

~1.2

and P20~-containing experiments with OCP and apatite seeds are shown in fig. 8. Points below each of the respective theoretical lines indicate solutions that are supersaturated with respect to that pure phase. Points above the theoretical lines represent solutions that are undersaturated with respect to that phase. It can be seen that the

(A) on OCP seeds

l~0 0.8

1.4 0

~

~apatlte

seeds

__________________________________ 0

10

100 Time (mm)

1000

10000

Fig. 6. The composition of the phase growing on OCP seeds (A) and on apatite seeds (B), as measured by the changes in ~Ca/~l P04 with time in the solutions. The ~1Ca/~~P04 values are the average of three independent analyses of Ca and P04. The corresponding standard deviations (error bars) are also shown,

solutions containing crystals growing on OCP seeds (fig. 8A) were supersaturated with respect to

OCP where the experimental line was below the theoretical line of OCP. Where the experimental

line crosses the theoretical line of OCP, the solution became undersaturated with respect to OCP but still supersaturated with respect to OHAp (fig. 8A). Hydrolysis of the phase that grew on the seeds should take place in the region where the

N. Eide!man et a!.

(A)

/

OCP

Selective inhibition of crystal growth on

649

and OHAp

on OCP seeds

34.0

33.0

° ° ______

—

—

—

experimental points regression

line

theoretical line: OCP theoretical line: OHAp

32.0

C

II)

~=L~TRO.990

31.0

_

30.0

(B) on apatite seeds 20

34.0

33.0

°

—

— —

4

10

22

‘

28

‘

34

(Degrees)

experimental points

Fig. 9. X-ray diffraction patterns: (A) OCP seeds; (B) OCP

regression line theoretical line: OCP

seeds plus the phase that grew on them in the presence of 1pM

theoretical

P

20~ (C) apatite seeds plus the phase that grew on them in the presence of 1pM P20~”;(D) apatite seeds.

line: OHAp

32.0

0~

31.0

the XRD patterns (figs.and 9B and 9C).seeds In fig.plus 10 the the FTIR spectra of OCP apatite

~

phases which grew on them in the presence of 30.0 16.2

b~.tMP20~ are shown. Here also it seems that the 16.4

16.6

16.8

17.0

spectrum of the original apatite seeds plus the

pCa(OH)z growing on OCP seeds (A) and on apatite seeds (B): data of

phase which grew on them in the presence of 1~tM P20~ (fig. bOC) is less sharp than the spectrum of

controls and 1pM P20~-containing experiments are under

the original seeds (fig. 1OD) (region 960—1100

Fig. 8. Chemical potential plots of the solutions of crystals

“experimental points”. The slope of the regression line for the growth on OCP seeds (A) is 1.29 (r = 0.990), and the regression line slope for growth on apatite seeds (B) is 1.26 (r

____________________________

=

0.998).

experimental line was between the theoretical lines of OCP and OHAp (fig. 8A). This is true also for the solutions containing apatite seeds (fig. 8B). In fig. 9 the XRD patterns of the OCP and apatite seeds and of the phases that grew on them in the presence of bfLM P20~ are shown. The pattern of

the phase that grew on the apatite seeds in the presence of lftM P20~ plus the seeds themselves (fig. 9C) reveals less developed apatite than the original seed crystals (fig. 9D) (region 20 = 31° to 34°). The pattern of the OCP seeds plus the overgrowth in the presence of 1~sMP20~ (fig. 9B) was almost as sharp as the pattern of the seeds

alone (fig. 9A). No additional reflections except these attributed to OCP or OHAp were found in

C.)

E I1-’

1500

I

1000

Wavenumbers(cm’)

500

Fig. FTIR spectra: (A) inOCP (B)of OCP seeds plus (C) the phase10.that grew on them the seeds; presence 1pM P20~”; apatite seeds plus the phase that grew on them in the presence of 1pM P20~’;(D) apatite seeds.

650

N. Eidelman ci al.

/ Selective

inhibition of crystal growth on OCP and OHAp

cm~). The librational band of the OH- at 631

shown in fig. 11. It can be seen that there was

cm appears only as a shoulder (fig. 1OC) and 1 (not the 0H stretching band at 3570 cm shown) appears to be smaller than this band in the original seeds. This spectrum also reveals small

some crystal growth (elevated titration, lower curve

bands of type-B carbonate (875, 1419, and 1457 cm i), The spectrum of OCP seeds plus the overgrowth resembles the spectrum of OCP by itself (figs. bOA and lOB). The observation that the XRD patterns and FTIR spectra of the OCP seeds plus the overgrowth of theexperiments control (not(figs. shown) 9B 74-containing and lOB) the P20 and resemble the pattern and spectrum of the original OCP seeds used (figs. 9A and bOA), indicating that the seeds did not hydrolyze significandy during the crystal growth experiments,

3.2.

Constant physiological P,O~- concentration

In order to mimic physiological conditions, an attempt was made to maintain a constant physiological P20~ concentration (ifiM) in supersaturated solution in contact with OCP seeds. This was done by repeated additions of labeled P2O~ to the solution until the level was quite constant (the total amount added was equal to 5.ljsM P2O~ if all had remained in solution). The results are

____________________________ 1.00

1.00

......./i 1

I

\

~

~

‘~a 0.75

1

[P2o7] I —A——KOH titration’

~-o---

h.~

0.50

0.25

0.00

~

0.50

—

0.25

~

0

10 Time

10~~ (mm)

10000

Fig. ii. OCP seeded crystal growth in nearly constant concentration of P20~. measured by 0.IN KOH titration with time. The lower curve is the titration curve for the growth on OCP seeds (tx), and the upper curve represents the concentrations of the labeled P20~ in the solution ~

in fig. 11) and some decrease in Ca concentrations (not shown) in the presence of a nearly constant b~sMP

2O~. The crystal growth in the presence of the constant physiological concentration of P20~ was slower than in the “one-time” addition experiments.

4. Discussion The present study shows that the rate of calcium phosphate crystal growth from metastable supersaturated solution was faster on the OCP seed crystals than on the apatite seed crystals in the presence of a “one-time” addition of the inhibitor, 1~M P 20~ (fig. 4). The result that the final titration volumes and concentrations of Ca and P04 in the control and P20~-containing experiments with both types of seeds were about the same after the induction time suggests that the same phase was growing on both seeds [24]. The high levels of the radioactive P2O~ found in the solid phases of the experiments with OCP seeds indicate that the P2O~ was incorporated into the crystal lattice or adsorbed strongly [24]. The inhibitory effect of the P20~ decreased with time (fig. 5), probably by adsorption on the crystals and also because of its hydrolysis to PO$ at the surface of OCP (this study and ref. [24]) and OHAp [2O,2b]. The crystal growth on the OCP seeds was accelerated when most of the P2O~ disappeared from the solution (fig. 5A), while in the same time period the growth on the apatite seeds was limited by the slow decrease of the P~0~in the solution (fig. SB). The results show that the Ca/P ratios the first day were 1.32 ±0.10 for the growth on the OCP seeds and 1.34 ±0.06 for the growth on the apatite seeds, increasing slowly thereafter to b.5 (fig. 7), .

.

suggesting that OCP (Ca/P = 1.33) precipitated first on both phases, followed by its hydrolysis to a more apatitic phase with a significantly lower .

Ca/P04 [24].

.

.

.

ratio than stoichiometric OHAp (1 .67)

N. Eide!man et a!.

/ Selective

inhibition of crystal growth on OCP and OHAp

651

The chemical potential plots show that the solubility of the phase that formed was much more consistent with OCP than with OHAp. The results from the XRD and the FTIR measurements that (a) the phase that grew on the apatite seeds in the presence of 1 1.tM P20~ plus the seeds themselves (figs. 9C and 1OC) is less developed apatite than the original seed crystals (figs. 9D and bOD), (b) the pattern of the OCP seeds plus the overgrowth (figs. 9B and lOB) was almost as sharp as the pattern of the seeds alone (figs. 9A and bA), and (c) no other phases of

lead directly to the formation of OCP crystals. These results are consistent with the OCP—precursor—hydrolyzate crystal growth mechanism proposed for bioapatite formation [4].

calcium phosphate except OCP or OHAp were

Israel, for his help in performing the FTIR mea-

found in the XRD patterns (fig. 9B and 9C) suggest also that probably OCP precipitated and

surements, and Dr. M.S. Tung for donating the OCP crystals.

hydrolyzed. The observation that the characteristic lines of OCP were not evident in the XRD

This investigation was supported, in part, by USPHS Research Grant HL30035 to the Ameri~

patterns and FTIR spectra of the apatite seeds plus the overgrowth does not preclude the presence of OCP as a precursor that hydrolyzed partially because OCP typically loses these peaks on partial hydrolysis and because the amount of the growing phase on the seeds was small relative to the amount of the original seeds. OCP is known as a labile compound that forms and hydrolyzes in solution under physiological conditions [1,7,19,29]; therefore, it can easily escape detection in biological systems [7,17]. The present study demonstrates that it is possible for some crystal growth to occur on OCP seeds in the presence of a constant physiological concentration of lfLM P2O~ (fig. bi, elevated titration with time), while another study [20] has indicated that crystal growth was not possible under similar conditions on OHAp seeds. It might be concluded from the present study that apparently OCP, but not OHAp, grew first on the OCP and apatite seeds in the presence of P20~ and then hydrolyzed to a more apatitic phase. This is consistent with the hypothesis that OCP is a precursor in formation of OHAp in vivo. In support of the present investigation is a study by Salimi et [36] that the shows that magnesium 2~)al.inhibited growth of OHAp

can Dental Association Health Foundation from the National Institutes of Health — National Heart, Lung, Blood Institute, and is part of the dental research program conducted by the National Institute of Standards and Technology in cooperation with the American Dental Association Health Foundation. Certain commercial materials and equipment are identified in this paper to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by the National Institute of Standards and Technology or the ADA Health Foundation or that the material or equipment identified is necessarily the best available for the purpose.

ions (Mg severely, of OCP moderately, and of DCPD very little. Mg2~,like P 20~, is known to inhibit growth of OHAp. However, it was not clear until recently [4,9] that inhibition of OHAp growth appears to

Acknowledgments

The authors would like to thank Dr. J.P. Cline,

Ceramics Division, NIST, for the use of the XRD, Dr. A. Givan,

Hebrew

University, Jerusalem,

References [1] WE. Brown, Nature (London) 196 (1962) 1048. [2] P.T. Cheng, Calcif. Tissue Intern. 37 (1985) 91. [3] F.C.M. Driessens, R.A. Terpstra, P. Bennema, J.H.M. Woeltgens and R.M.H. Verbeeck. Z. Naturforsch.

42c

(1987) 916. [4] WE. Brown, N. Eidelman and B. Tomazic, Advan. Dental Res. 1 (1987) 306. [5] Calcif. C. Siew, SE. Intern., Gruninger, L.C. Chow and WE. Brown, Tissue in press. [6] I. Mayumi and M. Yutaka, J. Crystal Growth 96 (1989)

[7] PT. Cheng, Calcif. Tissue Intern. 40 (1987) 339. [8] F.C. Driessens, G. Schaafsma, E.C. van Geresteijn and J. Rotgans, Z. Orthop. 124 (1986) 599.

652

N. Eidelman ci a!.

/

Selective inhibition of crystal growth on

[9] N. Eidelman, L.C. Chow and WE. Brown, Calcif. Tissue

Intern. 41(1987)18.

OCP and OHAp

[23] P.H. Cartier and L. Thuillier, Anal. Biochem. 61 (1974)

416.

[10] H.J. Hohling, R.W. Fearnhead and G. Lotter, Ger. Med. Monatsh. 13 (1968) 135.

[24] N. Eidelman, WE. Brown and J.L. Meyer. J. Crystal Growth 108 (1991) 385.

[11] D.J. McCarty, JR. Lehr and P.B. Halverson, Arthritis Rheum. 26 (1983) 1220. [12] H.E. Schroeder and H.U. Bambauer, Arch. Oral Biol. 11 (1986) 1. [13] CA. Saxton, Arch. Oral Biol. 13 (1968) 243. [14] J. Lustmann, J. Lewin-Epstein and A. Shteyer, Calcif. Tissue Res. 21(1976) 47. [15] HR. Schumacher, Jr., AL. Osterman, S.J. Choi and PB.

[25] J.L. Meyer and A.H. Reddi, Arch. Biochem. Biophys. 242

[17] R. Irby, W.M. Edwards and R. Gaiter, J. Rheumatol. 2 (1975) 91. [18] D. Caspi, 0. Rosenbach, M. Yaron, D.J. McCarty and E. Graff, J. Rheumatol. 15 (1988) 823. [19] R.Z. LeGeros, G. Daculsi, I. Orly, T. Abergas and W. Torres, Scanning Microsc. 3 (1989) 129. [20] J.L. Meyer, Arch. Biochem. Biophys. 231 (1984) 1.

(1985) 532. [26] N.S. Chickerur. MS. Tung and WE. Brown, Calcif. Tissue Intern. 32 (1980) 55. [27] N. Eidelman, L.C. Chow and WE. Brown, Calcif. Tissue Intern. 40 (1987) 71. [28] H. McDowell, TM. Gregory and WE. Brown, J. Res. Natl. Bur. Std. (US) 81A (1977) 273. [29] MS. Tung, N. Eidelman, B. Sieck and WE. Brown, J. Res. Nail. Bur. Std. (US) 93 (1988) 613. [30] Y. Avnimelech, E.C. Moreno and WE. Brown, J. Res. NatI. Bur. Std. (US) 77A (1973) 149. [31] G.L. Vogel, L.C. Chow and W.E. Brown, Caries Res. 17 (1983) 23. [32] J. Murphy and J.P. Riley, Anal. Chim. Acta 27 (1962) 31. [33] WE. Brown, in: Environmental Phosphorus Handbook, Eds. J. Griffith, A. Beeton, J.M. Spencer, D.T. Mitchell (Wiley, New York, 1973).

[21] H. Fleisch, R.G.G. Russell and F. Straumann, Nature (London) 212 (1966) 901. [22] R.G.G. Russell, S. Bisaz, A. Donath, D.B. Morgan and H. Fleisch, J. Clin. Invest. 50 (1971) 961.

[34] H.H. Ku, J. Res. Nail. Bur. Std. (US) 70C (1966) 263. [35] H.H. Ross, Anal. Chem. 41(1969)1260. [36] M.H. Salimi, J.C. Heughebaert and G.t-I. Nancollas, Langmuir 1(1985)119.

Weisz, Clin. Orthop. 219 (1987) 221. [16] L.G. Johnsson, R.C. Rouse, C.G. Wright, P.J. Henry and

i.E. Hawkins, Jr., Am. J. Otolaryngol. 3 (1982) 77.