Langmuir 1994,10, 4721-4725
4721
A Mineralization Adsorption and Mobility Study of Hydroxyapatite Surfaces in the Presence of Zinc and Magnesium Ions Tristan A. Fuierer, Marie LoRe,? Scott A. Puckett,* and George H. Nancollas* Chemistry Department, State University of New York at Buffalo, Buffalo, New York 14260 Received July 5, 1994@ Although relatively small amounts of zinc and magnesium ions are known to be incorporated into tooth enamel and bone minerals, they may have a major influence on the mineralization kinetics. In this study, the results of equilibrium adsorption of these ions on hydroxyapatite (HAP) and electrophoretic mobility measurements suggest that at solution concentrations of zinc ion above about 1ppm, the formation of zinc phosphate (Hopeite)dominates the surface properties. In the presence of zinc and magnesium ions, the constant composition growth of HAP is markedlyreduced and the results suggest a Langmuir-typeadsorption of these additives at active growth sites on the HAP crystals. Zinc ion is one of the most effective of the simple metal ions in inhibiting H A P crystal growth.
Introduction Studies of the influence of divalent metal ions such as magnesium and zinc on the mineralization kinetics of hydroxyapatite (HAP)are of importance in the elucidation of the mechanisms of calculus formation. The latter is composed primarily of calcium phosphate phases, most notably HAP,' and both magnesium and zinc ions reduce the rate of crystal growth of HAP when present in supersaturated s o l ~ t i o n s . ~Recently, J it has been shown that zinc ions and a phosphonate, 172-dihydroxy-1,2-bis(dihydroxyphosphony1)ethane)may synergistically inhibit HAP crystal g r ~ w t h .The ~ reduction of crystal growth rate in the presence of metal ions is usually attributed to the adsorption of the additives a t active growth sites on the HAP surfaces. Complexation ofmetal ions with lattice anions in the solution may also reduce the degree of supersaturation resulting in an apparent inhibitory e f f e ~ t . ~ Magnesium ion has been shown to stabilize the in vitro formation of amorphous or acidic calcium phosphate phases by preferentially inhibiting the crystal growth of the thermodynamically most stable HAP.596 It has also been reported that this cation stabilizes tricalcium phosphate in the precipitation of HAP.7 In vivo, the presence of magnesium ion extends the lifetime of amorphous calcium phosphate phases probably by reducing their effective solubilities.8 Despite the important roles played by both magnesium and zinc ions in the mediation of dental caries and calculus formation, most studies have been concerned with the former even though zinc ion, which has been shown to be present at levels from 430 to 2100 ppm in the surface of tooth enamel, may be much more effective in modifying t Present address, Calgon Corporation, Pittsburgh, PA 15230. Present address, School of Dentistry, University of Michigan, Ann Arbor, MI 48109. Abstract published inAdvance ACSAbstracts, October 15,1994. (1)Nikiforuk, G. Understanding Dental Calculus I Etiology and Mechanisms-Basic and Chemical Aspects; Karger: New York, 1985. (2)h a d , Z.; Koutsoukos, P.; Nancollas, G. H. J. Colloid Interface Sci. 1984,101, 250. (3)Dalpi, M.; Karayianni, E.; Koutsoukos, P. J.Chem.Soc.,Faraday Trans. 1993,89, 965. (4)Nancollas, G. H.;Tomazic, B.; Tomson, M. Croat. Chem. Acta 1976,48,431. (5)Brown, W.E.; Smith, J. P.; Lehr, J. R.; Frazier, A. W. Nature 1962,196,1050. (6)Tomazic, B.; Tomson, M.; Nancollas, G. H.Arch. Oral B i d . 1976, 20,803. (7) Newesely, H. Adu. Oral Biol. 1970,4,11. (8)Boskey, A. L.; Posner, A. S. Mater. Res. Bull. 1974,9, 907.
*
@
the mineralization kinetics. The smaller ionic radius of this ion, as compared with that of calcium, enables it to compete for cation positions in the calcium phosphate crystal l a t t i ~ e .In ~ addition to its marked influence on calculus formation, studies in vivo suggest that zinc salts hinder initial plaque formation and disrupt bacterial metabolism.lOJ1 Often included in antitartar dentifrice formulations, the effectiveness of zinc a s a n anticalculus agent is most probably due to a combination of a reduction of plaque growth, by interfering with its formation onto tooth surfaces, and the disruption of bacterial metabolism. Despite the inhibiting ability of magnesium on the rate of HAP crystal g r ~ u r t h , ~Neuman , ~ J ~ and Mulryan13 and Terpstra and Driessens14suggested that the ions are not incorporated in the apatite lattice to any great extent. LeGeros15 reported that magnesium substitution for calcium during apatite precipitation was limited to about 0.4 wt % and attributed a n observed decrease in apatite lattice parameters to magnesium incorporation. In contrast, Aoba et a1.16reported that 97% of the magnesium incorporated into synthetic apatite was present in crystalline lattice sites, but the experimental evidence was not conclusive. Since magnesium phosphates are more soluble than those of zinc, their inhibitory influence on HAP crystal growth can probably be interpreted in terms of surface adsorption rather than precipitation. In this study, the mineralization of HAP in the presence of zinc and magnesium ions has been investigated using the dual constant composition (DCC) method.17 The influence of these ions on the electrophoretic mobility of the HAP particles and equilibrium adsorption investigations were also made. In contrast to magnesium, the much lower solubility of zinc phosphate phases is a n important factor in controlling the effect of zinc ion on HAP (9)Brudevold, F.; Steadman, L. T.; Spinelli, A. A.; Amdur, B. H.; Gron, P. Arch. Oral Biol. 1963,8, 135. (10)Saxton, C. A.; Harrap, G. J.; Lloyd, A. M. J.Clin. Periodontal 1986,13, 301. (11)Jones, C. L.; Stephan, K. W.; Ritchie, J. A.; Huntington, E.; Saxton, C . A.; van der Oudera, F. J. Caries Res. 1988,22,84. (12)Feenstra, T. P.:. HOD, - . J.; . deBruyn, - . P. J. J. Colloid Interface Sci. 1981,83,583. (13)Neuman, M. F.; Mulryan, B. J. Calcif. Tissue Res. 1971,7,133. (14)Terpstra, R.A.; Driessens, F. C. Calcif. Tissue Int. 1988,39, 348. (15)LeGeros, R. Z. In Tooth Enamel N; Fearnhead, S., Ed.; Elsevier: Amsterdam, 1984;pp 32-36. (16)Aoba, T.; Moreno, E. C.; Shimoda, S. Calcif. Tissue Int. 1992,51, 143. ~. (17)Ebrahimpour, A.;Zhang, J.; Nancollas, G. H. J. Cryst. Growth 1991,113, 83. ~
0743-7463/94/2410-4721$04.50/00 1994 American Chemical Society
4722 Langmuir, Vol. 10, No. 12, 1994
Fuierer et al.
Table 1. Uptake of Zinc Ions at HAP Surfaces from Solutions Saturated with HAP initial Tznf mol L-l 0.700 1.22 1.84 2.44 3.06 4.60 15.3 30.6 45.9 61.1 76.5
equil Tzd 10-4 mol L-1
aopeite
0.0067 0.070 0.280 0.583 1.15 2.11 2.04 4.79 7.50 10.1 13.1
-0.229 0.0747 0.374 0.626 0.861 1.37 3.80 6.13 7.91 9.39 10.7
solution was raised slowly t o the required value by the addition of 0.01 mol L-l potassium hydroxide solution. Crystal growth was induced by the addition of approximately 20 mg of HAP seed crystals. Calcium (Orion 932000) and pH glass (Orion 910100) ion specific electrodes were used to control the addition of titrant solutions A and B, respectively,in order to maintain the constancy of ionic activities in the supersaturated solutions. The concentrations of titrant solutions, Ti,are expressed in terms of the supersaturated solution concentrations, Wi, in eq 2
titrant A
TC,= 2Wc,
mineralization. Marked differences in the nucleating properties of HAP surfaces pretreated with magnesium and zinc ions were of special interest in light of the importance of these additives in dental applications.
+ 5Ce,
titrant B: Tp = 2Wp + 3Ce,
Materials and Methods Analytical grade chemicals and triply distilled carbon dioxidefree water were used for the preparation of solutions. Zinc, magnesium, and calcium ion concentrations were determined by EDTA complexometric titrations, by atomic absorption spectroscopy, (Perkin-ElmerModel 31001, and/or by inductively coupled plasma emission spectroscopy(Therm0 Jarrell Ash ICAF' Model 61). Phosphate concentrations were determined spectrophotometrically (Varian Cary 210 spectrophotometer) as the vanadomolybdate phosphate complex.'e H A P seed crystals were prepared using a modification of the method described previously.19 Titration of 2.0 L of 0.15 mol L-1 (NHdzHP04 in 1.5mol L-l NH40H into 7.0 L of boiling 0.08 mol L-l Ca(NO& in 0.2 mol L-l N&OH was completed over a period of 5h. This was followed by the addition of 0.060 L of concentrated N K O H to maintain the pH a t 10. The solution was refluxed for 12 h and the precipitate was filtered and washed with triply distilled water to remove excess supernatant solution ions. The washed precipitate was resuspended in 0.15 mol L-l NaCl and aged for at least 3 months at 37 "C. The molar ca/po4ratio was 1.67 f 0.01 and the specific surface area was 26.3 f 0.3 m2 g-l (BET nitrogen adsorption; 30170 N a e Quantasorb, Quantachrome Corp.). X-ray diffraction analysis (Nicolemic with STOE attachment) also confirmed the phase as HAP. The equilibrium adsorption of zinc ions a t HAP surfaces was studied over a range of concentrations with hopeite relative supersaturations in the solutions, u, ranging from -0.22 t o 10.7 where u = [IP/K,,l""
-1
(1)
In eq 1,IP is the hopeite ionic product [(Zn2+)3(P043-)2], K,,the solubility product, and Y is the number of ions in the formula unit. Adsorption measurements on known masses of HAP seed (usually approximately 1.0g L-l) in solutions of ZnClz were made in 0.15 mol L-l NaCl and equilibration was allowed to proceed a t 37 "C for 4 h before centrifugation a t 2000 rpm for 10 min and supernatant solution analysis. This equilibration time was shownto be at least twice that required for completeequilibration. Magnesium ion adsorption experiments were made using a similar technique. The solid phases were subjected to electrophoretic mobility measurements using a Malvern Zetasizer IIc following redispersion in saturated H A P solution a t pH values of 7.0 or 7.4, with ultrasonification for about 5 min to break up crystal aggregates. Crystallization kinetics experiments were made in doublewalled Pyrex glass vessels maintained at 37 "C. Supersaturated solutions (0.200 L) were prepared by the careful mixingofsodium chloride, potassium dihydrogen phosphate, and calcium chloride solutions while bubbling with water vapor saturated nitrogen gas in order to exclude carbon dioxide. After the addition of zinc or magnesium chloride solutions, the pH of the supersaturated (18)Tomson, M. B.; Barone, J. P.; Nancollas, G. H. At. Abs. Newsl. 1977,16,117. (19)Ebrahimpour, A. Ph.D. Dissertation, S U N Y a t Buffalo, 1990.
In eq 2, P represents phosphate and Ces,the effective concentration of titrant solutions with respect to HAP. This was chosen mol L-l so as to provide a conveniently measurable a t 1.00 x rate oftitrant addition. During the crystallization experiments, samples were withdrawn periodically, filtered (0.22,um Millipore filters), and analyzed to verify constancy of calcium and phosphate concentrations. In addition, solid samples were examined by scanning electron microscopy (Hitachi S800 field emission microscope). Constant composition crystal growth experiments were made in the presence ofmagnesium and zinc ions at pH 7.0 with U H A = ~ 5.22. Crystal growth was initiated by the addition of HAP seed or HAP seed preadsorbed, for 4 h a t 37 "C, with zinc or magnesium.
Results and Discussion The ionic products for calculating the crystallization driving force (eq 1)must be expressed in terms of free ion activities. These were calculated using expressions for mass balance, electroneutrality, and equilibrium constants for phosphate protonation and for the association of these ions with the divalent cations. Values of the association constants were as follows: ZnH2P04+, 39.81 L mol-l; ZnHP04,1995 L mol-'; CaH2P04+,28.1 L mol-l; CaHP04, 589 L mol-'; MgHP04, 741 L m01-1.20-23 Activity coefficients were calculated from the extended form of the Debye-Huckel equation proposed by DaviesVz4 Adsorption and mineralization experimental conditions are summarized in Tables 1 and 2, respectively. The uptake of zinc ions a t the HAP surface is plotted as a function of equilibrium concentration of adsorbate in Figure 1. In the region of low zinc concentrations, the initial high affinity uptake is followed by a plateau and can be interpreted in terms of a Langmuir isotherm, expressed as eq 3 CIQ = 1lKN
+ CIN
(3)
In this equation, C is the equilibrium concentration of adsorbate, Q the amount adsorbed, Kthe affinity constant, and N the maximum concentration (saturation) of adsorption sites.25 The plateau a t low Zn concentration (20) Zhang, J.; Ebrahimpour, A.; Nancollas, G. H. J. S o h . Chem. 1991.20. 455.~ . . .--, (21) Tabor, H.; Hastings, A. B. J. Biol. Chem. 1943,148, 627. (22)Childs,C. W. I n o g . Chem. 1970,9,2465. (23) Nriagu, J. 0. Geochim. Cosmochim. Acta 1973,37,2357. (24) Davies, C. W. Ion Association; Butterworths: London, 1962; p ~
190. (25) Kresak, M.; Moreno, E. C.; Zahradnik, R. T.; Hay, D. I. J.Colloid Interface SOC.1977,59, 283.
Mineralization Adsorption of HAP
Lungmuir, Vol. 10, No. 12, 1994 4723
Table 2. Crystal Growth Experiments at 37 "C and I = 0.16 mol L-' in NaCl expt no. 12 27O 17a 2w 22a 29 117 168 169 171 172 133
~ ~ ~ 1 1 0 - 4 TP/~O-4 mol L-1 mol L-1 4.00 2.40 4.00 2.40 4.00 2.40 4.00 2.40 4.00 2.40 4.00 2.40 10.0 6.00 10.0 6.00 10.0 6.00 10.0 6.00 10.0 6.00 10.0 6.00
PH 7.40 7.40 7.40 7.40 7.40 7.40 7.00 7.00 7.00 7.00 7.00 7.00
TzJ~O-~ mol L-l
R&O-* mol
TM&O-~ mol L-1
UHAP
0
3.60 3.60 3.60 3.60 3.60 3.60 5.22 5.22 5.22 5.21 5.20 5.10
3.82 5.00 7.65 15.3 5.00 0
0.990 2.50 9.90 15.0 99.0
min-l
'Jhopeite
0
-0.216 -0.078 0.189 0.802 -0.078
m-2
2.4 0.53 0.50 0.43 0.33 0.40 31.7 15.4 12.5 11.3 9.27 2.21
Greater than 95% zinc uptake by HAP seed. ~ , ~ / i o - ' m o i L-' 0.0
10,
0.5
1.0
1.5
2.0
,
2.5
25
20
c)
I
3
15
gb
e
10
-P Y
5
-1.50
I
0
0 0
3
6
9
12
15
10
5
1 .o
0.5
I
,
8
12
15
20
adsorbed/lO-' mol m-' Figure 3. Electrophoretic mobility of HAP, preadsorbed with zinc ions at pH 7.4. Zn
15
C*~/lO-"ol L-l Figure 1. Zinc adsorption isotherms on HAP at lower (filled circles) and higher (open circles) concentrations.
0 0.0
0
4
1.5
c z ~ / i O - ' m ~ iL-'
Figure 2. Langmuir isotherm of zinc at low concentrations.
suggests a n effective monolayer coverage and a typical plot of eq 3 is shown in Figure 2, with calculated Kzn = 1.5 x lo5 L molw1and N = 7.7 x 10-6-mol m-2. The adsorption curves at higher zinc concentrations and pH = 7.0 in Figure 1 (open circles) are essentially linear at these relatively high supersaturations with respect to hopeite (Table l), suggesting the formation of hopeite at the HAP surfaces. In order to further investigate the behavior of HAP surfaces in the presence of zinc ion, electrophoretic mobility measurements were made at pH 7.4. The data are plotted in Figure 3. In the absence of additive, the HAP mobility was 0.27 f 0.02 pm s-l V-' cm-l and, following the uptake of zinc ions, the mobility would be expected to become more positive. However, it is seen that this increase in mobility occurs only at very low concentrations of adsorbed zinc ion (less than 0.02 mg
m-9. As the concentration of additive increases, there was a dramatic reduction in mobility to an approximately constant value of -1.3 pm s-l V-l cm-l. It is significant that this mobility value is close to that determined experimentally for synthetic hopeite under the same conditions.26 The decrease in mobility of HAP in the presence of zinc ions thus appears to be accounted for by the formation of negatively charged zinc phosphate at the surface. Moreover, multilayering of hopeite probably occurred as indicated by the constant HAP mobility over a n extensive range of zinc concentrations. Since the magnesium phosphates are much more soluble than the corresponding zinc salts, the formation of these phases a t HAP surfaces is unlikely. The adsorption isotherm for magnesium onto HAP, shown in Figure 4, indicates that maximum adsorption is not attained in the concentration range studied. In contrast to zinc, the uptake of magnesium ions at HAP crystal surfaces results in the expected increases in electrophoretic mobility shown in Figure 5. Moreover the monotonic increase in mobility with increasing magnesium ion concentration, in contrast to the limiting hopeite value in the case of zinc, also suggests multilayering of magnesium ion species a t the surface. The results of crystal growth experiments a t pH 7.4 are summarized in Table 2 and typical plots of titrant volume as a function of time are shown in Figure 6. I t can be seen that the presence of zinc results in a marked inhibition of crystal growth. To reduce the possibility of concomitant hopeite precipitation, low concentrations of zinc ion were used in these experiments and the concentrations of supersaturated solutions were adjusted so as to maintain a value f&opit, = 0. The rate of crystal growth was obtained (26) Elder, B. J. personal communication,
SUNY
at Buffalo, 1993.
4724 Langmuir, Vol. 10, No. 12, 1994
%
E
,
14
-
12
-
10
-
E
B -
B 7
6 -
>
4 -
3
0
Fuierer et al. Y
I
I
*
0
v -
0
1
3
2
4
7
6
5
0
5
L-'
15
20
Figure 7. Rate of HAP growth plotted against concentration of zinc ions at pH 7.4.
Figure 4. Adsorption isotherm of magnesium onto HAP. 0.15 0.10
3'0
0.05
f
10
Conc. Zn/tO-' mol L-'
2.5
* 1 -1
0.00
> l o
-0.05
0)
$ 2
-0.15
cl .* 3
-0.20
-
'
-O.'O
-0.25
3
0
6
9
15
12
0.0
Figure 5. Electrophoretic mobility of HAP, preadsorbed with magnesium ions at pH 7.0. 14
1 l 20
'
0
Mg adsorbed/iO-'mol m-*
I/
600
400
800
1000
time/min
Figure 8. Moles of HAP grown plotted against time at pH 7.4: 0, expt 17; 0, expt 29, HAP growth with aliquot of zinc (5.00 x mol L-l) added at 250 minutes.
c
t
If it is assumed that zinc ions adsorb a t kink growth sites on the HAP surfaces, the surface concentration may be expressed as a relative reduction in growth rate in terms of a Langmuir-type isotherm (eq 5).
Expt. 12
+
JdJi = 1 K'C,
0
4
1
200
':
1.
0
200
400
600
800
1000
time/min
Figure 6. Plots of titrant volume against time. Dashed line is titrant A (Ca Na Zn), solid line is titrant B (OH PO&
+ +
+
from the titrant volume data using eq 4
J g = (dV+jdt)C,@SA
m
(4)
in which Vt is the volume of titrant added, SSA is the specific surface area, and m is the mass of initiating seed crystals. In Figure 6 it can be seen that a zinc concentramol L-l was sufficient to almost tion of 1.53 x completely inhibit crystal growth. The addition of zinc ion in the range 3.8 x to 15.3 x mol L-l (Figure 7) reduced the growth rate of HAP by 78% to 86%. Chemical analysis of filtered aliquots removed during the DCC experiments verified a concentration constancy to within f2% for both calcium and phosphate ions.
(5)
In eq 5, JOand Ji are the growth rates in the absence and presence of additive, respectively, and Ci is the total additive c o n ~ e n t r a t i o n .The ~ ~ derived "kinetic" affinity constant,K'z, = 2.1 x lo5L mol-' compares satisfactorily with the value 1.5 x lo5 L mol-l, obtained from the adsorption experiments. In Table 2, it can be seen from the data for experiments 17,20,22, and 27 that there was a greater than 95% uptake of zinc ions from solution by the H A P seed crystal surfaces. It is not possible to conclude whether this represents adsorption or calcium ion replacement since if zinc had replaced lattice calcium ions, the change in solution concentration of the latter would be less than 1%. The speed a t which the growth is inhibited is illustrated in Figure 8 (experiment 29). In this case, once a steady HAP crystal growth rate had been achieved, the system was spiked with zinc ion (5.0 x mol L-l). It can be seen that the reduction in growth rate was observed within a few minutes. Comparison with experiment 17, in which a similar concentration of zinc ion was present in the supersaturated solution, (Table 2) shows satisfactory agreement in the final growth rates. (27) Christoffersen,J.; Christoffersen,M. R. J.Cryst. Growth 1981, 53, 42.
Langmuir, Vol. 10, No. 12, 1994 4725
Mineralization Adsorption of HAP 10,
0
20
40
00
60
Conc. Mg/lO-'mol
100
L-'
Figure 9. Rate of crystal growth in the presence of magnesium ions at pH 7.0.
It is interesting to note that Amjad et a1.2 derived a "kinetic" affinity constant, R M=~ 1.54x lo4 L mol-l, a t pH 7.40,37 "C, and U H A = ~ 8.65. The inhibitory influence of magnesium ion in solution at U H A= ~ 5.22and pH 7.0is shown in Figure 9 and Table 2. The addition of magnesium ion in the concentration range (0.990to 99.0)x mol L-' reduced the growth rate of HAP by 51% to 93%. In view of the relatively high solubility of magnesium phosphate species, HAP crystal growth inhibition probably occurred by adsorption of magnesium ions at active growth sites. However, this ion was a much less effective inhibitor of HAP crystal growth than zinc since about 24 ppm magnesium was required to produce 90% inhibition as compared with only about 1 ppm zinc ion. HAP crystallization results of experiments in which magnesium and zinc ions were preadsorbed on the seed crystals are shown in Figure 10. As the concentration of preadsorbed magnesium ion increased, the rate of HAP growth decreased monotonically. In the case of zinc, however, preadsorbed concentrations of 5.1 x and
0
,
I
50
100
150 200 time/min
250
300
350
Figure 10. A comparison of the inhibitory effects of preadsorbed magnesium and zinc at pH 7.0,conditionsas in expt 117 (Table 2): 0, no additive; 0, 5.1 x mol L-l zinc; 0, 4.5 x mol L-l zinc; A, 1.3 x mol L-l magnesium; A, 1.3 x mol L-l magnesium.
4.5 x mol L-l resulted in almost the same degree of HAP inhibition suggesting that the lower concentration was sufficient to effectively block all growth sites. Preadsorbed zinc was therefore much more effective in inhibiting crystal growth than preadsorbed magnesium ions. This may be due to not only the formation of zinc phosphate complexes but also the concomitant partial hydrolysis of the surface zinc species enabling them to more effectively block multiple active growth sites. In contrast, the magnesium ion will exist primarily as the free cation in solution perhaps limiting the adsorption potential. Acknowledgment. We thank the National Institutes of Health for a grant (DE03223)in support of this work.
