Langmuir 2000, 16, 6745-6749
6745
Inhibition of Hydroxyapatite Crystal Growth by Substituted Titanocenes S. Koutsopoulos, E. Dalas,* and N. Klouras Department of Chemistry, University of Patras, GR-26500 Patras, Greece Received January 20, 2000. In Final Form: May 17, 2000 This paper reports a physicochemical investigation of the effect of titanocenes on hydroxyapatite crystallization in vitro. The experimental conditions resemble the physiological, at sustained supersaturation employing the constant composition method. All titanocene dihalides were found to inhibit crystal growth of hydroxyapatite, possibly through adsorption of the chloride hydrolysis products on the active growth sites. It is suggested that the growth mechanism is probably surface-diffusion-controlled spiral growth. To explain the inhibiting activity of the titanocenes, the relation of the organometallic compound stereochemistry and the crystal surface characteristics is extensively discussed.
Introduction For a long period of time, there was little scientific interest in doing basic research on the pharmaceutical use of organometallic compounds. This was basically due to the poisoning action of heavy metals, and it was not assumed that such metals could have therapeutic efficiency in coordinating surroundings. The first studies on the relationship between the chemical structure and therapeutic efficacy or toxicity of organometallic compounds began only about 60 years ago.1 Over the past 50 years, a large amount of work has been done on metallocene dihalides. It has been shown that these compounds, of the form R2MX2 (where R ) η5-C5H5, η5-CH3C5H4, etc.; M ) Ti, Zr, Hf, and V; and X ) F, Cl, Br, and I), are highly effective against Ehrlich ascites tumor cells, sarcoma 180, B16 melanoma, lymphoid leukemia L1210, lymphocytic leukemia P388, and colon 38 carcinoma.2-4 The effectiveness of metallocenes as antitumor agents varies with the position of the metal atom in the periodic table.5 The carcinostatic activity of metallocenes is mechanistically similar to that other agents such as cisplatin, (NH3)2PtCl2, targeting the nucleic acid metabolism and mitotic function.6-8 Titanocene dihalides have been clinically tested in the past, and they proved to be drastic therapeutic drugs against arthritis.9,10 Recently, much work has been done in testing these compounds, and physicochemical evidence for the antiarthritic activity of several metallocene complexes has been reported.9-15 * Corresponding author. (1) Collier, W. A.; Krauss, F. Z. Krebsforsch. 1931, 34, 527. (2) Ko¨pf-Maier, P.; Hesse, B.; Voitgtla¨nder, R.; Ko¨pf, H. J. Cancer Res. Clin. Oncol. 1980, 97, 31. (3) Ko¨pf-Maier, P.; Wagner, W.; Hesse, B.; Ko¨pf, H. Eur. J. Cancer. 1981, 17(6), 665. (4) Ko¨pf-Maier, P.; Wagner, W.; Ko¨pf, H. Cancer Chemother. Pharmacol. 1981, 5, 237. (5) Ko¨pf-Maier, P. In Metal Complexes in Cancer Chemotherapy; Keppler, B. K., Ed.; VCH: Weinheim, Germany, 1993; pp 261-296. (6) Ko¨pf-Maier, P.; Ko¨pf, H. Naturwissenschaften 1980, 67, 415. (7) Ko¨pf-Maier, P.; Wagner, W.; Ko¨pf, H. Naturwissenschaften 1981, 68, 272. (8) Rosenberg, B.; van Camp, L.; Trosko, J. E.; Mansour, V. H. Nature 1969, 222, 385. (9) Fairlie, D. P.; Whitehouse, M. W.; Broomhead, J. A. Chem.-Biol. Interact. 1987, 61, 277. (10) Simon, T. M.; Kunishima, D. H.; Vibert, G. J.; Lobert, A. Cancer Res. 1981, 41, 94. (11) Dalas, E.; Klouras, N.; Maniatis, C. Langmuir 1992, 8, 1003.
Hydroxyapatite [HAP, Ca5(PO4)3OH] is thermodynamically the most stable calcium phosphate salt and is the inorganic component of hard tissues such as bones and teeth.16-20 On the other hand, HAP formation occurs in several pathological cases of undesirable biological calcification. Articular cartilage calcification, atheromatic plaque, renal calculi, and the formation of bladder and bile stones are a few examples of its involvement.21-26 Thus, HAP has been considered as the model compound for the in vitro study of biomineralization processes. In the present work, the effect of the titanocene dihalides listed in Table 2 was investigated. Their chemical structure can be seen in Figure 1. For the kinetic study of hydroxyapatite crystal growth, the constant composition method was employed.27-29 Experimental Section The titanocene dihalides were prepared and purified according to methods reported in the literature.30-33 They were characterized by elemental analysis (C, H, and X), which gave deviations (12) Koutsopoulos, S.; Demakopoulos, I.; Argiriou, X.; Dalas, E.; Klouras, N.; Spanos, N. Langmuir 1995, 11, 1831. (13) Koutsopoulos, S.; Dalas, E.; Tzavellas, N.; Klouras, N. J. Chem. Soc., Faraday Trans. 1997, 93 (23), 4183. (14) Koutsopoulos, S.; Dalas, E.; Tzavellas, N.; Klouras, N.; Amoratis, P. J. Cryst. Growth 1998, 183, 251. (15) Koutsopoulos, S.; Dalas, E. J. Mater. Sci., Lett. 1998, 17, 485. (16) Katcburian, E. J. Anatomy 1973, 116, 285. (17) Dalas, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1989, 85, 2465. (18) Neuman, W. F.; Neuman, M. W. Chemical Dynamics of Bone Mineral; The University Press: Chicago, IL, 1958; p 67. (19) Neuman, W. F. Bone Material and Calcification Mechanisms. In Fundamental and Clinical Bone Physiology; Urist, M. R., Ed.; J. B. Lippincott: Philadelphia, PA, 1980; p 83. (20) Christoffersen, J.; Christoffersen, M. R. J. Cryst. Growth 1981, 53, 42. (21) Nancollas, G. H. J. Cryst. Growth 1977, 42, 185. (22) Boskey, A. L.; Bullogh, P. G. Scan. Electron Microsc. 1984, 28, 511. (23) Gordon, G. V.; Villanueva, T.; Shumacher, H. R.; Gohel, V. J. Rheumatology 1984, 11, 861. (24) Valente, M.; Bortloti, U.; Thiene, G. Am. J. Pathol. 1985, 119, 12. (25) Scho¨en, F. J.; Levy, R. J. Cardiol. Clin. 1984, 2, 713. (26) Koutsopoulos, S.; Kontogeorgou, A.; Petroheilos, J.; Dalas, E. J. Mater. Sci.: Mater. Med. 1998, 9, 421. (27) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. Soc. 1980, 102, 1553. (28) Tomson, M. B.; Nancollas, G. H. Science 1978, 200, 1059. (29) Koutsoukos, P. G. Ph.D. Thesis, State University of New York at Buffalo, Buffalo, NY, 1980. (30) Reynolds, L.; Wilkinson, G. J. Inorg. Nucl. Chem. 1959, 9, 86.
10.1021/la000070j CCC: $19.00 © 2000 American Chemical Society Published on Web 07/06/2000
6746
Langmuir, Vol. 16, No. 16, 2000
Figure 1. Titanocene dihalides, complexes with a tilted sandwich structure. of e0.5%, and the infrared spectra, X-ray crystallography data, and 1H NMR spectra showed no evidence of impurities. For the kinetic experiments, grade A glassware and triply distilled, CO2-free water were used to prepare stock solutions from the respective solids (analytical reagent chemicals, Merck). All solutions were filtered before use (0.22-µm Millipore filters). The filters were prewashed to remove residuals and surfactants. For standardization of the stock solutions, the concentrations of phosphate and calcium ions were determined by spectrophotometry as the vanadomolybdate complex34 and by ion-chromatography (Metrohm 690-Ion Chromatography Detector, IC Y-521 Shodex), respectively. Potassium hydroxide solutions were prepared from concentrated standards (Titrisol, Merck). The crystal growth experiments were performed in a covered double-walled, water-circulated cell with a volume totaling 250 mL that was thermostated at 37 ( 0.1 °C. One hour before the start of the experiment, working solutions were prepared after the introduction of the titanocenes into phosphate solutions containing the HAP seed crystals at the desired pH and ionic strength by the addition of the appropriate volume of calcium chloride solution. This procedure is advantageous compared to the classical constant composition technique because of the time allowed for the solid crystal substrate to interact with the diluted additive foreign complex. The ionic strength and pH of the solutions were adjusted to 0.15 M and 7.4 by the addition of sodium chloride and potassium hydroxide, respectively. Before and during the crystallization process, water-presaturated nitrogen gas was bubbled through the solution to exclude dissolved atmospheric CO2. The HAP seed crystals were prepared by a method described elsewhere.35 They were analyzed by infrared spectroscopy (KBr pellet method, FT-IR Perkin-Elmer 16-PC), powder X-ray diffraction (Philips PW 1830/1840, Cu KR radiation), and chemical analysis. The specific surface area of the crystals, as determined by a triple-point BET method (PerkinElmer Sorptometer 212 D), was found to be 34.6 m2 g-1. The synthetic crystals displayed the characteristic powder X-ray diffraction pattern36 and the infrared spectrum of stoichiometric HAP.29 The experimentally determined molecular ratio of calcium to phosphate (Ca:P) in the solid material was 1.67 ( 0.04. The reaction process was monitored through the pH change in the working solution. The pH was measured using a combined glass/Ag/AgCl electrode (Metrohm, 6.0202.100), standardized before and after each experiment with NBS standard buffer solutions.37 The HAP precipitation resulted in a lowering of the pH, which originated from the protons released from the reacted KH2PO4 and the KOH depletion (because of the phosphate and hydroxyl groups incorporation into the HAP crystal lattice). A pH drop of 0.005 pH units triggered the addition of reactants from two mechanically coupled burets mounted on a modified pH-stat apparatus (Metrohm 614, Impulsomat). The titrant solution concentrations are given in detail elsewhere.38 Samples (31) Wilkinson, G.; Birmingham, J. M. J. Am. Chem. Soc. 1954, 76, 4281. (32) Samuel, E. Bull. Soc. Chim. Fr. 1966, 11, 3584. (33) Ko¨pf, H.; Klouras, N. Chem. Ser. 1982, 19, 122. (34) Tomson, M. B.; Barone, J. P.; Nancollas, G. H. At. Absorpt. Newsl. 1977, 16, 1179. (35) Amjad, Z.; Koutsoukos, P. G.; Nancollas, G. H. J. Colloid Interface Sci. 1984, 101, 250. (36) ASTM File Card No. 9-432; American Society for Testing and Materials: West Conshohocken, PA. (37) Bates, R. G. Determination of pH. Theory and Practice; Wiley: New York, 1973.
Koutsopoulos et al. were taken randomly during the course of the reaction, filtered (0.2-µm Millipore), and analyzed for calcium and phosphate, thus verifying the constancy in the concentration as better than 3%. The precipitates were also analyzed using X-ray powder diffraction, scanning electron microscopy, and chemical analysis to ensure that no phase transformationoccurred. The crystal growth rate (i.e., moles of HAP formed per minute and square meter of surface area of the substrate) was determined from the volume of titrant added per time unit. The reproducibility of the measured growth rates was better than 6%. Experiments with different amounts of seed crystals (10, 15, and 20 mg) gave the same initial rates normalized per unit surface area of the substrate. It can, therefore, be suggested that crystallization took place exclusively on the surface of the introduced seed crystals.39 Higher amounts of seed crystals led to erroneous results because, at the working ionic strength (i.e., 0.15 M NaCl), particle aggregation occurred. Also, changes in the stirring rate between 100 and 800 rpm had no effect on the growth rates (R), suggesting that the rate-determining step of the process is not bulk diffusion from the bulk solution to the crystal surface.40 Crystal seed particles in the working solution had a mean size of 10 µm, as measured with a particle size analyzer (Laser Particle Counter ILI-1000-SPECTREX).
Results and Discussion The solution speciation in the crystal growth experiments was calculated from the proton dissociation and ion pair formation constants for calcium and phosphate, the mass balance, and the electroneutrality conditions by successive approximations of the ionic strength.41 The activity coefficients were computed using the extended Debye-Hu¨ckel formula proposed by Davies.42 At 37 °C, the thermodynamic solubility products of the calcium phosphate salts are, for hydroxyapatite (HAP), k°s ) 2.35 × 10-59; 43 for tricalcium phosphate (TCP), k°s ) 2.83 × 10-30;44 for octacalcium phosphate (OCP), k°s ) 5.01 × 10-50;45 and for dicalcium phosphate dihydrate (DCPD), k°s ) 1.87 × 10-7.46 The driving force for the formation of z+ zXu, where u ) (u+) + (u-), is the a crystalline phase Mu+ change in Gibbs free energy, ∆G, for the transfer from the supersaturated solution to equilibrium.
∆G ) -RgT ln
RgT IP (Mz+)u+(Xz-)uln )(1) k°s u k°s
In eq 1, parentheses denote activities, IP is the ionic product of the precipitating salt, k°s is the solubility product, u for HAP is equal to 9, Rg is the gas constant, and T is the absolute temperature. The experimental conditions, thermodynamic data, and kinetic results obtained are summarized in Tables 1 and 2. In all cases, except with the F- counterion, the presence of the titanocene compound suppressed the rates of HAP crystallization. The titanocene complexes were active even at concentration levels of 10-5 mol dm-3, inhibiting the rate of HAP crystal growth to an extent of 50%. The rate of crystal growth was also reduced upon increasing the (38) Koutsopoulos, S.; Paschalakis, P. C.; Dalas, E. Langmuir 1995, 10, 2423. (39) Ny´vlt, J.; So¨hnel, O.; Matuchova´, M.; Broul, M. The Kinetics of Industrial Crystallization; Elsevier: Amsterdam, 1985; pp 68, 284. (40) Nielsen, A. E.; Toft, J. M. J. Cryst. Growth 1984, 67, 278. (41) Nancollas, G. H. Interactions in Electrolyte Solutions; Elsevier: Amsterdam, 1966. (42) Davies, C. W. Ion Association; Butterworth: London, 1962. (43) McDowel, H.; Gregory, T. M.; Brown, W. E. J. Res. Natl. Bur. Stand. 1977, 81A, 273. (44) Gregory, T. M.; Moreno. E. C.; Patel, J. M.; Brown, W. E. J. Res. Natl. Bur. Stand. 1974, 78A, 667. (45) Moreno, E. C.; Browm, W. E.; Osborn, G. Soil Surf. Proc. 1960, 99. (46) Marchall, R. Ph.D. Thesis, State University of New York at Buffalo, Buffalo, NY, 1970.
Inhibition of Hydroxyapatite Crystal Growth
Langmuir, Vol. 16, No. 16, 2000 6747
Table 1. Crystallization of HAP on HAP Seed Crystals in the Presence of (MeC5H4)2TiCl2a ∆G (J/mol) TCP OCP
exp. no.
(MeC5H4)2TiCl2 (10-5 mol/L)
Cat (10-4 mol/L)
HAP
S-315 S-175 S-176 S-174 S-173 S-177 S-178 S-179R S-179β S-179γ
0.00 1.78 3.56 5.33 8.89 17.78 3.56 3.56 3.56 3.56
5 5 5 5 5 5 4 3.5 3 2.5
-4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -4.43 × 103 -3.94 × 103 -3.63 × 103 -3.29 × 103 -2.87 × 103
-3.99 × 102 -3.99 × 102 -3.99 × 102 -3.99 × 102 -3.99 × 102 -3.99 × 102 1.63 × 102 5.00 × 102 8.90 × 102 1.35 × 103
1.28 × 101 1.28 × 101 1.28 × 101 1.28 × 101 1.28 × 101 1.28× 101 5.00 × 102 7.99 × 102 1.14 × 103 1.55 × 103
DCPD
R (10-8 mol min-1 m-2)
3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 3.56 × 103 4.11 × 103 4.45 × 103 4.85 × 103 5.31 × 103
11.30 5.92 4.96 3.55 2.90 2.64 4.28 3.99 2.76 2.17
a Conditions: pH 7.40, 0.15 M NaCl, and Ca :P ) 1.67, where Ca and P are the total calcium and total phophate concentrations, t t t t respectively.
Table 2. Crystallization of HAP on HAP Seed Crystals in the Presence of Substituted Titanocene Dihalide Complexes (RC5H4)2TiX2a exp. no.
titanoceneb (3.5 × 10-5 mol/L)
R (10-8 mol min-1 m-2)
c
Cp2TiCl2
S-Ti-1 S-32-OM S-33-OM S-34-OM
(Me3SiC5H4)CpTiF2 (Me3SiC5H4)CpTiCl2 (Me3SiC5H4)CpTiBr2 (Me3SiC5H4)CpTiI2
11.80 5.35 5.55 5.16
S-35-OM S-36-OM S-37-OM S-38-OM
(MeC5H4)CpTiF2 (MeC5H4)CpTiCl2 (MeC5H4)CpTiBr2 (MeC5H4)CpTiI2
12.10 4.33 4.09 4.66
S-39-OM
(Me3SiC5H4)2TiCl2
5.89
S-40-OM
Cp2TiS5
1.78
S-115 S-41-OM S-42-OM
(Et3SiC5H4)2TiF2 (Et3SiC5H4)2TiCl2 (Et3SiC5H4)2TiBr2
S-176 S-112
(MeC5H4)2TiCl2 (MeC5H4)2TiBr2
2.00
Figure 2. Kinetics of HAP crystallization both in the presence (b) and in the absence (2) of 3.56 × 10-5 mol dm-3 (MeC5H4)2TiCl2 at pH 7.40, 37 °C, and 0.15 mol × dm-3 NaCl.
13.07 7.00 6.19 5.83 5.85
a Conditions: pH 7.40, 0.15 M NaCl, Ca ) 5 × 10-4 M, Ca :P t t t ) 1.67, where Cat and Pt are the total calcium and total phophate b concentrations, respectively, and ∆GHAP ) -4.43 kJ/mol. Me ) -CH3, Et ) -CH2CH3. c Data from ref 11.
solution concentration of the metallocene, as shown in the detailed study of the effect of (MeC5H4)2TiCl2 against HAP crystallization (Table 1). The relative solution supersaturation, σ, is defined as
σ)S-1)
( ) IP k°s
1/9
-1
(2)
which, for HAP, takes the form
σ)
3 - 1/9 [(Ca2+)5(PO3- (k°s)1/9 4 ) (OH )]
(k°s)1/9
-1
(3)
where S is the solution supersaturation. The relative solution supersaturation was found to influence the rate of HAP crystallization, as can be seen from the experimental results in Table 1. The dependence of the relative solution supersaturation on the crystal growth rate, R, of HAP can be described by the phenomenological equation
R ) ksσ
n
(4)
where k is the crystallization rate constant, s is a function of the active growth sites on the surface of the seed crystals, and n is the apparent order of the crystal growth reaction. Kinetic plots according to eq 4 gave a satisfactory fit, as
can be seen in Figure 2. From the linear plots, values of n ) 1.7 ( 0.2 and n ) 1.9 ( 0.1 were obtained for the crystallization of HAP in the presence and in the absence of (MeC5H4)2TiCl2, respectively. This kinetic results imply that the crystal growth mechanism is not affected by the presence of the titanocene dichloride. A value of n ≈ 2 is indicative of a surface-diffusion-controlled spiral growth mechanism.47-50 According to the literature the inhibitory effect of a soluble compound can be attributed to one or more of the following factors: (i) The additive forms stable complexes with one or more of the reactant ions thus, reducing the solution supersaturation. (ii) The inhibitor may change the ionic strength of the working solution, affecting both the supersaturation level and the solubility of the precipitating salt. (iii) The solute interacts with the crystal surface and adsorbs on it entropically (on a free surface) or in energetically favored active growth sites (e.g., kinks and steps). The inhibition observed cannot be ascribed to a decrease in the solution supersaturation because of complexation of the Ca2+ with the titanocenes. This was shown by potentiometric titrations of the titanocene complexes at the experimental conditions both in the presence and in the absence of a total calcium concentration of 5 × 10-4 M. Random sampling during the precipitation reaction and chemical analysis for calcium and phosphate verified (47) Nancollas, G. H. In Biomineralization; Mann, S., Webb, J., Williams, R. J. P., Eds.; VCH Verlagsgesellschaft: Weinheim, Germany, 1989; Chapter 6, pp 156-187. (48) Dalas, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1 1989, 85, 2465. (49) Maniatis, Ch.; Dalas, E.; Zafiropoulos, Th.; Koutsoukos, P. G. Langmuir 1991, 7, 1542. (50) Dalpi, M.; Karayianni, E.; Koutsoukos, P. G. J. Chem. Soc., Faraday Trans. 1993, 89 (6), 965.
6748
Langmuir, Vol. 16, No. 16, 2000
Koutsopoulos et al. Table 3. Affinity Constants for Various Inhibitors of HAP Crystal Growth
Figure 3. Kinetics of HAP crystal growth in the presence of various concentrations of (MeC5H4)2TiCl2 according to the Langmuir model at pH 7.40, 37 °C, and 0.15 mol × dm-3 NaCl.
the constancy of the solution supersaturation. Also, potentiometric titrations at the experimental conditions of the titanocene complexes both in the presence and in the absence of a total phosphate concentration of 3 × 10-4 M did not show appreciable complexation of phosphate ions. This can be explained by the fact that the titanocene concentration in supersaturated solution was 1 order of magnitude less than the phosphate concentration. The reduction in the crystal growth rate of HAP in the presence of titanocene dihalides cannot be explained in terms of an increase in the ionic strength, because the concentration of the titanocene is 6 orders of magnitude lower than the concentration of the supporting electrolyte (i.e., 0.15 M NaCl). Therefore, there is no decrease in the supersaturation level of the precipitating solution due to change of the ionic strength. Thus, the inhibition observed can only be ascribed to further blocking of the active growth sites of the seed crystals. Fitting the kinetic results in a modified Langmuir-type isotherm tested this assumption. It should be noted, however, that the basic assumptions on which the kinetic isotherm is based are that the adsorption free energy is constant over the entire adsorbent surface and that there are no lateral interactions between the adsorbed molecules. The rates of crystal growth in the absence, Ro, and in the presence, Ri, of the inhibitors can be related to the inhibitor concentration in the supersaturated solutions, Ci, according to
kd 1 Ro )1+ Ro - Ri ka Ci
(5)
where ka and kd are the specific rate constants for adsorption and desorption, respectively. The ratio (ka/kd), defined as the affinity constant, Kaff, can be determined from plots of Ro/(Ro - Ri) against 1/Ci, according to eq 5, as in the plot shown in Figure 3. The excellent linearity of the plot and the intercept, which is equal to unity, as predicted from eq 5, are strong indications for the validity of the model and the assumptions made. From the slope of the straight line, a value of 5.55 × 104 dm3 mol-1 was obtained for the affinity constant of (MeC5H4)2TiCl2. For comparison, values of affinity constants of other metallocenes for the surface of HAP are provided in Table 3. A higher value of the affinity constant indicates stronger adsorption of an inhibitor on the HAP surface. As can be seen from Table 3, the (MeC5H4)2TiCl2 complex has a lower affinity constant for the crystal surface of HAP compared to that of the unsubstituted (C5H5)2TiCl2. This can be explained if the hydrolysis products stereochemistry is considered. The volume of the substituted compound is much larger. because of the presence
inhibitor
104 (ka/kd) dm3 mol-1
ref
titanocenes [Cp2Ti(H2O)2]2+ zirconocenes [Cp2Zr(H2O)2]2+ Vanadocenes [Cp2V(H2O)2]2+ hafnocenes [Cp2Hf(H2O)2]2+ titanocenes [MeC5H4)2Ti(H2O)2]2+
68.9 57.8 11.9 10.0 5.55
11 12 13 14 this work
of the cyclopentadienyl-ring ligand, than the volume of the latter, and so, the adsorption procedure for the first is more difficult, for stereochemical reasons, than that of the second. This fact is reflected in the inhibitory effects of the two compounds (Table 2). It is important to note that the inhibitory effect of the titanocenes was not followed by a calcium phosphate phase transformation, as demonstrated by the precipitate analysis (X-ray powder diffraction pattern, FT-IR spectrum, and chemical analysis identical with those of the stoichiometric HAP). Titanocenes resist cyclopentadienyl ring loss. At 37 °C, in solutions 0.103 M in NaCl, there was no detectable ring loss after 2 days.51 Therefore, the amount of free cyclopentadiene in the working solution 3-8 h after the introduction of the titanocene dihalide was insignificant. Chloride hydrolysis gives the following products in the working solution according to the reactions:51
(RC5H4)2TiCl2 + H2O ) [(RC5H4)2Ti(H2O)Cl]+ + Cl(6) (RC5H4)2Ti(H2O)Cl+ + H2O ) [(RC5H4)2Ti(H2O)2]2+ + Cl- (7) The chloride hydrolysis procedure proceeds quite rapidly (the first step cannot be measured by chloride potensiometry, whereas for the second, the half-life is nearly 0.5 h at 37 °C and 0.318 M KNO3).51 The creation of positively charged molecules by chloride hydrolysis results in a strong electrostatic interaction between the negatively charged HAP crystal surface12 and the positive [(RC5H4)2Ti(H2O)Cl]+ and [(RC5H4)2Ti(H2O)2]2+ complexes, thereby resulting in an expected strong adsorption on the crystal surface and further blocking of the active growth sites. Aside from (MeC5H4)2TiCl2, the effects of other titanocene dihalides on HAP crystallization kinetics are presented in Table 2. Their activities depend on the halide and the nature of the organic group bound to the cyclopentadienyl ring of the titanocene. It is important to note that titanocene difluorides accelerated the initial crystal growth rate of HAP. This observation is controversial, given the general inhibiting behavior of the other metallocene dihalides.11-14 This accelerating phenomenon is attributed to the F- ions, produced from the hydrolysis reactions (eq 6 and 7).52,53 Similar results with other compounds containing fluoride ions have also been reported in the literature.11 Fluoride ions in concentrations such as 1 ppm are capable of accelerating HAP formation and enchancing reminerallization of the enamel surface.54,55 The results can also be interpreted via possible Ca5(PO4)3F (FAP) formation, because of the presence of fluoride ions, F-, in the supersaturated solutions (the solutions are supersaturated with respect to this phase). (51) Toney, J. H.; Marks, T. J. J. Am. Chem. Soc. 1985, 107 (4), 947. (52) Amjad, Z.; Koutsoukos, P. G.; Nancollas, G. H. J. Colloid Interface Sci. 1984, 101 (1), 250. (53) Nancollas, G. H.; Tomson, M. B. Faraday Discuss. Chem. Soc. 1976, 61, 175.
Inhibition of Hydroxyapatite Crystal Growth
Langmuir, Vol. 16, No. 16, 2000 6749
FAP is less soluble than HAP, and both have the same crystallographic characteristics (i.e., the same crystal symmetry and almost the same crystal lattice parameters).54 The formation of FAP has also been found to be kinetically favored among calcium phosphate salts, and therefore, it precipitates faster than HAP from supersaturated calcium phosphate solutions.52,55 FAP crystals were not detected with the available analytical means because it was formed at negligible quantities. In titanocene dichlorides, the situation is clearer because only the inhibiting action of the additive in the crystallizing solution is of concern. In the present case, it is important to study the products and the kinetics of the hydrolysis steps (eqs 5 and 6). The solute species from the disintegration of the organometallic complex interact with the active growth sites on the HAP crystal surface. As can be seen from Table 2, (Et3SiC5H4)2TiCl2 was found to be a weaker inhibitor of HAP crystal growth as compared with the other titanocene dichlorides tested. This can be attributed to the nature of the Et3Si- organic group, which is an electron donor to the cyclopentadienyl ring. The presence of the Et3Si- group in the complex results in stabilization of the titanocene against further hydrolysis and delocalizes the positive charge, which appears on the Ti atom after chloride hydrolysis. Furthermore, because of its hydrophobic characteristics and its bulky stereochemical configuration, (Et3SiC5H4)2TiCl2 resists the hydrolysis of the bond between the substituted cyclopentadienyl group and the central atom. The (Me3SiC5H4)2TiCl2 complex has the same behavior against HAP crystallization. This reflects the similarity between the Me3Si- group bonded to the cyclopentadienyl ring and the Et3Si- group of the previous complex. (Me3SiC5H4)2TiCl2, on the other hand, is a weaker inhibitor than the nonsymmetric (Me3SiC5H4)CpTiCl2, in which one of the cyclopentadienyl rings has no side organic group. It is also noteworthy to mention that titanocene dichlorides with at least one unsubstituted cyclopentadienyl group (e.g., Cp2TiCl2, (Me3SiC5H4)CpTiCl2, and (Et3SiC5H4)CpTiCl2) are stronger inhibitors than the symmetrical forms, in which both aromatic rings have the same side group. This observation points to the fact that the adsorption process of the solute titanocenes on the crystal surface, which results in the inhibition of HAP crystal growth, is favored when the aromatic ring is free from substitution, especially if the substitute is large. Thus, it can be suggested that the substituted cyclopendadienyl
group influences the positive charge of the chloride hydrolysis products (eq 6 and 7). As far as the titanocene dibromides are concerned, the complexes with symmetrical substitution on the cyclopentadienyl ring, (Et3 SiC5H4)2TiBr2 and (MeC5H4)TiBr2, have a less inhibiting effect against HAP crystal growth than the nonsymmetrical complexes. The same observation was made for the dichlorides as well. The same holds for the case of the titanocene diiodides, and the results are presented in Table 2. It is also important to mention the inhibitory effect of Cp2TiS5, which retards the crystal growrth of HAP to an extent of 84%. In this compound, a six-membered heterocyclic ring TiS5 is formed. The introduction of Cp2TiS5 in the environment of the supersaturated solution probably results in the formation of complex ions of the type [Cp2Ti(H2O)]2+ through hydrolysis and further sulfur-ring disintegration, as in analogous complexes.5,56 Thus, the similarity of the inhibiting activity of Cp2TiCl2 and Cp2TiS5 can be explained (Table 2). Furthermore, there is an additional inhibiting effect of the sulfur ions, which have often been accused of poisoning the crystal growth sites on the crystal surface.57 Another possibility is the cyclopentadienyl protonolysis, which takes place in organometallic complexes51 with different kinetics mechanisms for any one of them.
(54) Amjad, Z.; Koutsoukos, P. G.; Nancollas, G. H. J. Dent. Res. 1981, 60 (10), 1783. (55) Amjad, Z.; Koutsoukos, P. G. and nancollas, G. H. J. Dent. Res. 1982, 61 (9), 1094.
(56) Dombrowski, K. E.; Baldwin, W. and Sheats, J. E. J. Organomet.Chem. 1986, 302, 281. (57) Lindsay, W. L. Chemical Equilibria in Soils; Wiley: New York, 1979; pp 281-298.
Conclusions In the present work, the effect of titanocene complexes on the crystallization of HAP was investigated at a constant composition solution supersaturated only with respect to HAP. The titanocene dihalides were shown to be strong inhibitors, reducing the rates of crystal growth of HAP by 54-94%. The additives were found to be significantly active against HAP crystallization at concentration levels of 10-5 mol dm-3. The crystal growth mechanism, as revealed by kinetic data, was probably surface-diffusion-controlled spiral growth. The adsorption and subsequent blocking of the active growth sites by the chloride hydrolysis products may explain the inhibitory effect. The adsorption assumption may be justified through the satisfactory fit of the results to a kinetic Langmuirtype model. Acknowledgment. We acknowledge the partial support of the Ministry of Energy and Technology of the Greek Government, Grant E∆ 857. LA000070J
