Crystallization of Hydroxyapatite on Oxadiazole-Based Homopolymers

Crystal Growth & Design , 2006, 6 (6), pp 1547–1552. DOI: 10.1021/cg060015u. Publication Date (Web): April 26, 2006. Copyright © 2006 American Chem...
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Crystallization of Hydroxyapatite on Oxadiazole-Based Homopolymers J. Kanakis, A. Chrissanthopoulos, N. P. Tzanetos, J. K. Kallitsis, and E. Dalas* Department of Chemistry, UniVersity of Patras, GR-26504 Patras, Greece

CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 6 1547-1552

ReceiVed January 10, 2006; ReVised Manuscript ReceiVed March 27, 2006

ABSTRACT: Oxadiazole homopolymer was found to be a substrate favoring the deposition of hydroxyapatite (HAP) crystals from stable supersaturated solutions at pH 7.40, 37 °C, and 0.15 M NaCl. The second-order dependence of the precipitation of HAP on oxadiazole polymer on the solution supersaturation suggested a surface diffusion controlled mechanism. The surface energy of the HAP nuclei growing on oxadiazole homopolymer was calculated to be 158 mJ m-2 from the dependence of crystal growth rate on the solution supersaturation. The overgrowth of HAP on the polymer was done selectively possibly through active site formation on -N-N- groups on the macromolecules as confirmed by computational chemistry calculations at 310 K. Introduction The deposition of calcium phosphate salts on polymers is of paramount importance not only for fundamental research concerning biomineralization1-4 but also for practical application, such as the design and development of new materials suitable for prosthetic applications in bone and teeth.5-7 So far, structural aspects of the polymer substrates employed and their relationship to the overgrowth calcium phosphate phase have been considered for the explanation of observed cases of deposition of these salts on polymers.8-10 In supersaturated solutions with respect to calcium phosphate salts, a number of calcium phosphate phases may be formed in order of increasing solubility: hydroxyapatite (Ca5(PO4)3OH, HAP), tricalcium phosphate (b-Ca3(PO4)2, TCP), octacalcium phosphate (Ca4H(PO4)3‚2.5H2O, OCP), anhydrous dicalcium phosphate (CaHPO4, DCPA), and dicalcium phosphate dihydrate (CaHPO4‚2H2O), DCPD). TCP and DCPA are higher temperature products, never reported to form spontaneously from solutions at ambient temperatures. It should be noted that biological fluids are supersaturated with respect to HAP only, due to extensive complexation of calcium with biological macromolecules.11 As a result free calcium concentration levels are low, thus precluding the formation of any phase other than HAP, and the in vitro experiments should be conducted at low supersaturations.12,13 In the present work, we have used oxadiazole homopolymers14,15 as nucleators of HAP. The main reason for our choice of those polymers was their outstanding mechanical and chemical properties, which would make them serious candidates for implants. The -N-N- units of oxadiazole not only offer the possibility of having a more favorable polymeric conformation but also provide nucleation sites for HAP through Ca2+ ion accumulation.10,16 The evaluation of the polymers employed for HAP nucleation and growth was done by the constant superaturation approach,17-19 which is unique for the accuracy and reproducibility it offers in kinetic measurements of crystal growth from aqueous solutions. Experimental Section A. Preparation of the Homopolymers Used as Substrates. Materials. 1-(4-Vinylbenzyloxy)-3,5-bis[5-(phenyl)-2-oxadiazolyl]benzene (I) (Scheme 1) was synthesized according to known procedures.1,2 * Corresponding author. Tel: +302610-997145. Fax: +302610-997-118. E-mail address: [email protected]

Azobis(isobutyronitrile) (AIBN) was recrystallized from methanol and stored in the freezer. All other chemicals and solvents were used as received from Aldrich. The polymerization reaction was carried out under an argon atmosphere. Instrumentation. IR spectra were recorded on a Perkin-Elmer 16PC FT-IR spectrometer with KBr pellets. 1H NMR spectroscopy measurements were performed using a Bruker Avance DPX 400 MHz spectrometer. Molecular weights (Mn and Mw) were determined by gel permeation chromatography using two Ultrastyragel columns of 500 and 104 Å pore sizes, respectively, a UV detector, CHCl3 as eluent (analytical grade), which was filtered through a 0.5 µm Millipore filter, a flow rate of 1 mL/min at room temperature and polystyrene standards for calibration. The sample was passed through a 0.2 µm Millipore filter. The UV-visible absorption spectra were recorded on a HewlettPackard 8452 A diode array UV-visible spectrophotometer. Free-Radical Polymerization of Monomer I. A solution of oxadiazole monomer I (1.50 g, 3.01 mmol) and AIBN (14.8 mg, 3% mol of monomer) in dry DMF (15 mL) was degassed three times and flushed with argon. The mixture was heated under stirring in a sealed tube for 6 days. After the mixture cooled to room temperature, CHCl3 (10-15 mL) was added to the reaction mixture to dissolve the polymer. The polymer was precipitated in a large excess of methanol (20-fold excess by volume) and further purified by reprecipitation from CHCl3 into ethyl acetate. Thus the obtained white solid was dried under vacuum at room temperature. Yield 0.408 g (27%). 1H NMR (CDCl3): 1.251.7 (two broad, CH2CH); 5.20 (broad, OCH2); 6.58-7.14 (broad, oxadiazole-CArH); 7.18-7.59 (broad, oxadiazole-CArH); 7.59-8.18 (broad, oxadiazole-CArH). Monomer and Polymer Synthesis. Scheme 1 shows the chemical structures of the monomer and oxadiazole-based homopolymer synthesized in this study. Free radical polymerization with AIBN as the initiator was utilized for the polymerization of the monomer I (Scheme 1). The unreacted monomer was effectively removed using dissolution of the reaction mixture and precipitation in a good solvent for the monomer (ethyl acetate for the monomer I). Resulting polymer P exhibited good solubility in a wide range of organic solvents, including CHCl3, THF, and DMF, which might be primarily due to the oxadiazole-based side groups. The above polymer was characterized by means of 1H NMR and UV-vis spectroscopies. The polymer P, in CHCl3 solution, exhibits an absorption maximum at 280 nm, which is the absorption region of the oxadiazole moiety (not shown here). The homogeneity of the polymer was assessed by gel permeation chromatography based on calibration with polystyrene standards, revealing a Mn of 7600 and a polydispersity of 1.63. Chromatogram was recorded at the standard 254 nm (detects aromatic nuclei). Gel permeation chromatography was also used to make sure that no traces of unreacted monomer remained in the homopolymer. As shown in Figure 1, GPC does not reveal residual monomer. The homopolymers were obtained as powders and were characterized by infrared spectroscopy (Perkin-Elmer 16-PC FT-IR using KBr pellets), powder X-ray diffraction (Phillips, 1300/00, Cu KR radiation), scanning electron microscopy (JEOL JSM 5200) with an energy-dispersive X-ray

10.1021/cg060015u CCC: $33.50 © 2006 American Chemical Society Published on Web 04/26/2006

1548 Crystal Growth & Design, Vol. 6, No. 6, 2006

Kanakis et al. Scheme 1a


(i) AIBN, DMF.

Figure 1. GPC chromatogram of the polymer in CHCl3 detected at 254 nm. microprobe (EDXS), and LEO supra 35 VP thermogravimetric analysis and differential scanning calorimetry (Du Pont 10 system coupled with a 990 programmer recorder). The specific surface area of the polymer powder determined by nitrogen adsorption multiple point BET (PerkinElmer model 212D sorptometer) was 47 m2 g-1. It is well-known that such polymers with metal-complexing units in the side chains adsorb irreversible cations from the bulk solution.7,9,10,14,15 For this reason, the polymer powder was suspended in 0.1 mol dm-3 calcium chloride solution for 24 h under continuous stirring. Next it was washed with 1 L of distilled water. Analysis for the washings showed no calcium desporption. Preequilibration with calcium was necessary for avoiding any possible alteration of the solution supersaturation, caused by Ca2+ uptake, which under our experimental conditions was negligible, as confirmed from chemical analysis of the supersaturated solutions. B. Crystallization Experiments. All experiments were done at 37.0 ( 0.1 °C in a 0.250 dm3 double-walled reactor thermostated with circulating water from a water bath. Stock solution of calcium nitrate and potassium dihydrogen phosphate were prepared from solid reagents (Merck, proanalysis) using triply distilled, CO2-free water. Standardization was carried out as described elsewhere.17 Prior to standardization, all solutions were filtered through membrane filters (0.22 µm Millipore). The supersaturated solutions were prepared in the glass reactor by mixing equal volumes (0.1 dm3 each) of calcium and phosphate solutions. The solution pH was adjusted by the slow addition of standard potassium hydroxide under nitrogen atmosphere, which was ensured by bubbling water-vapor-saturated prepurified nitrogen (Linde Hellas) through the supersaturated solution. All experiments were conducted at pH 7.40 and ionic strength 0.15 M, adjusted by NaCl. Following pH adjustment, all solutions in this work were stable at least for 3 days, as indicated from the constancy of pH and of the solution composition. After a waiting period of 4 h for each experiment, a quantity of 50 mg of powdered homopolymers was introduced in the supersaturated solution. The powder was thoroughly dispersed in the

magnetically stirred supersaturated solutions. It should be noted that the preequilibrium of the polymers with the 0.1 mol dm-3 calcium chloride solutions did not have any effect on their capability of inducing HAP overgrowth. In preliminary experiments, it was verified that polymers introduced in the supersaturated solutions without any pretreatment induced HAP precipitation as well, and the same relatively low induction times were observed. Preequilibration was considered necessary considering the limited ion binding capacity of the -NN- containing polymer, which however was not effectively demonstrated in our experimental conditions. The growth of HAP on oxadiazole homopolymer was preceded by relatively low induction times (less than five minutes), corresponding to the process of the formation of the HAP nuclei, which may further growth by a crystal growth process. These low values of the induction times observed preceded the formation of supercritical heterogeneous nuclei and depended on the initial calcium concentration. Similar results were observed in cases of HAP formation on fibrin6 or HAP precipitation on collagen type I19 where the overgrowth of HAP started without any appreciable induction time. Because of the low value induction times observed, thus producing significant experimental error, we adopt the rates of crystal growth at time zero for further kinetic treatment. The precipitation process, accompanied with proton release, triggered the addition of calcium chloride, potassium dihydrogen phosphate, and potassium hydroxide titrants from a pH-stat, modified in a way to allow the solution supersaturation to be kept constant.17-19 The precipitation process was monitored by a glass/saturated calomel pair of electrodes (Metrohm) standardized with NBS standard buffer solutions, while periodical analysis (every 30 min) of samples withdrawn from the working solution confirmed the constancy of calcium and phosphate concentrations throughout the course of precipitation within 2%. The solution supersaturation was thus kept constant, and the rate of titrant addition recorded as shown in Figure 2 gave directly a measure of the rate RG (at time zero) of calcium phosphate overgrowth on the polymer substrate. In the present work, the rates of crystal growth at the time of the onset of precipitation were used for the kinetic treatment to avoid taking into account the fact that the HAP crystals formed may serve as seed crystals accelerating the rate of precipitation. Any changes in the Ca/P molar ratio from 1.67 of the precipitated calcium phosphate lead directly in dramatic changes in solution composition, and the pH state goes out of control.17 The above referred in the literature in the case of TCP precipitation on ceramic phosphates,5 OCP on fabrin,6 and OCP on collagen type I.19 Furthermore in the experiments presented herein, the constancy of calcium and phosphate concentrations throughout the course of precipitation was tested by atomic adsorption spectrophotometry (Varian 1200) for calcium and for phosphate, as vanadomolybdate complex spectrophotometrically at 420 nm (Varian, Cary 219). The solid phases formed during the course of precipitation were examined by FT-IR spectroscopy, powder X-ray diffraction (at t ) 20 min from the beginning of the experiment), scanning electron microscopy, and thermogravimetric analysis.

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Figure 2. Crystal growth of HAP on oxadiazole homopolymer: plots of the volume of titrant addition as a function of time. Table 1. Crystallization of HAP on Oxadiazole Homopolymer, pH 7.40, 37 °C, 0.15 M NaCl, at Varying Total Calcium and [Ca]t/ (Total Phosphate) for [P]t ) 1.67 [Ca]t(10-4 mol dm-3)


5 4.5 4 3.5

3.56 3.84 4.12 4.45

∆G (kJ mol-1) OCP TCP


RG (10-8 mol min-1 m-2)


-3.99 -1.18 1.63 5.04

-4.43 -4.18 -3.94 -3.63

4.3 ( 0.13 3.7 ( 0.07 2.9 ( 0.04 1.8 ( 0.02

15.48 14.62 13.77 12.69

1.28 3.16 5.04 7.99

Results and Discussion The overgrowth of HAP on the water-dispersed oxadiazole homopolymers started without any appreciable induction time (very small induction times observed, less than 5 min). The experimental conditions are summarized in Table 1. Ionic speciation in solution was calculated from the proton dissociation and ion pair formation constants for calcium and phosphate, the mass balance, and electronutrality conditions by successive approximation for the ionic strength.17 The driving force of the calcium phosphate polymorph formation is the change in Gibbs free energy, ∆G, for the transfer from the supersaturated solution to equilibrium

∆G )

-RgT IP ln o V K

In eq 1, IP is the ionic product of precipitating salt, Kos its solubility product, V the number of ions (V ) 9 for HAP), Rg the gas constant, and T the absolute temperature. The following values were used for the thermodynamic solubility products of the various calcium phosphates: for HAP, Kos ) 4.7 × 10-59,20 for TCP, Kos ) 1.2 × 10-29,21 for OCP, Kos ) 8.3 × 10-48 23 (value obtained from ref 22 after correction for ion pair formation), and for DCPD, Kos ) 2.49 × 10-7.23 The relative solution supersaturation, σ, is defined in eq 2 as

[(Ca2+)5(PO43-)3(OH)]1/9 - (Kos )1/9 (Kos )1/9


(IP)1/9 - (Kos )1/9 (Kos )1/9

HAP21 and was found to strongly influence the rate of HAP precipitation RG (at time zero), as may be seen from Table 1. The BCF approach brought contemporary physics to crystal growth theory and stimulated further development of crystal growth science.24 At the experimental conditions (with relatively low supersaturation level) described in this work, the spiral growth model introduced by Burton-Cabrera-Frank (BCF) theory was found to give better interpretation of the kinetic data.25 According to the Burton, Cabrera, Frank theory law for crystal growth,26 the relationship between the rates and the relative supersaturation is given by

k gσ 2 B RG ) tanh B σ



where kg is the crystallization rate constant and B a kinetic constant dependent on diffusion and temperature.27 For low σ values (σ , B),

RG ) k′gσ2


And for relatively high σ values,




Figure 3. Plot of the rate of precipitation of HAP (at time zero) on oxadiazole homopolymer as a function of the relative solution supersaturation, pH 7.40, 37 °C, 0.15 M NaCl.

) Ω1/9 - 1 (2)

where parentheses denote activities, Ω is the supersaturation ratio, and Kos is the thermodynamic solubility product of

RG ) k′′gσ


The experimental data in the present work were fitted according to eq 4 with a value k′ ) 1.53 × 10-9 as shown in Figure 3. The second-order dependence for the HAP crystal growth has been found in the literature.5,6,9,10,28 It would be noted that the rates of HAP overgrowth on oxadiazole homopolymers measured were found to depend on the surface area of the substrate and that they were doubled or halved using twice or half the amount of the oxadiazole polymer powder used to initiate precipitation in an experiment. It may therefore be suggested that the process is surface diffusion controlled. Stoichiometric analysis of the inorganic phases showed a stoichiometry of Ca/P ) 1.67 ( 0.02, which is consistent with the solution concentrations of calcium and phosphate, which remained constant throughout the precipitation process. Small deviations from the stoichiometric coefficients of the growing crystalline phase would cause significant alterations in the solution composition. Moreover the exclusive formation of HAP was confirmed (a) by powder X-ray diffraction analysis29 [exhibits the characteristic reflections for HAP with d spacing 3.440, 2.817, 2.79,

1550 Crystal Growth & Design, Vol. 6, No. 6, 2006

Figure 4. Powder X-ray diffraction spectrum of HAP grown on oxadiazole homopolymer (t ) 120 min), [Ca]t ) 5 × 10-4 M, 37 °C, pH ) 7.40, 0.15 M NaCl.

Figure 5. Scanning electron micrographs of HAP grown on oxadiazole homopolymer (t ) 120 min), [Ca]t ) 5 × 10-4 M, 37 °C, pH ) 7.40, 0.15 M NaCl.

2.723, 2.265 and h k l (002), (211), (112), (300), (130), respectively] as shown in Figure 4, (b) by FT-IR spectroscopy,6,19,28 (c) by elemental analysis, and (d) by scanning electron microscopy. Morphological examination of the precipitated solid on oxadiazole homopolymer revealed the formation of HAP19,28 shown in Figure 5. The nucleating capability of macromolecules may be correlated with the possibility of binding of metal ions (e.g., Ca2+) with specific functional groups30,31 initiating the formation of subcritical nuclei, which grow to the critical size needed for

Kanakis et al.

crystal growth. Thus polymers with certain molecular weight and at defined concentration ranges may act as catalysts of the crystal growth process.32-35 In the case of oxadiazole homopolymer, we tried to assess the ion binding and the initialization of the crystallization process via computational chemistry calculations at 310 K, using for simplification of the calculations and minimization of the computer time, the Mg2+ ion instead of Ca2+ assuming similar behavior with the phosphate anions.36,37 The above was corroborated by the fact that the incorporation of Mg2+ in the bulk structure of sparingly soluble salts instead of Ca2+ has almost the same energetic content.38 Also in the computational model, no water molecules were taking into account to reduce the calculating time and the size of the model.39 The semiempirical level of molecular orbital (MO) theory, developed under the Hartee-Fock theory using various approximations,40 provides a useful tool to understand and predict the chemical properties of moderate size molecules. The calculations were performed with the parametric method 3 (PM3)41 included in version 6.0 of the MOPAC program package,42 because this is the only one having parameters for all used atoms (C, H, O, N, P, Mg). This method is based on the neglect of diatomic differential overlap (NDDO) formalism, employs an s-p basis set, and does not include d orbitals. It has been reported43 that it has mean unsigned errors in molecular geometries of 0.036 Å (bond lengths), 3.93° (angles), and 14.9° (torsion angles). The geometries were fully optimized in Cartesian coordinates (XYZ) using the BFGS method or the eigenvector following routine (EF). The final geometry corresponds to a stationary point on the potential surface, and this is one of many possible conformations. The aim of this study was to investigate the possibility of an amino acid chain binding magnesium hydroxy phosphate molecules and to find the active sites. To apply a MO method to macromolecules such as polymers, we simulate the long chain polymeric structure by finite models, oligomers having terminal hydrogens. The ability of these oligomers to bind magnesium hydroxy phosphates has been studied. The geometries were fully optimized in Cartesian coordinates (XYZ) using the BFGS method or the eigenvector following routine (EF). The final geometry corresponds to a stationary point on the potential surface, and this is one of many possible conformations. First we optimize the structure of the oligomer model, and after that magnesium hydroxy phosphate species with a Mg/PO4/OH stoichiometry equal to 2:1:1 and 5:3:1 were introduced at a distance of about 4 Å from the chain, and the system was let free to find its equilibrium position. Many starting geometries have been used to find any possible active site on the chain. The most active site is the dinitrogen (-NN-) unit of the polymer chain. Three to four magnesium atoms are located at a distance of about 2.5 Å, and the rest have been found at distances higher than 4 Å from the dinitrogen group (see Figure 6). The order of the N‚‚‚Mg bond is in the range 0.10-0.15.The distorted tetrahedral phosphate units are situated near magnesium and bound to it via one, two, or three oxygens. Hydrogen bonds between oxygen of the phosphates and hydrogen of the chain have been observed. The above results support the idea that the complexation of Ca2+ at the -N-Nunits of the chain is possible and that the complexes formed can initiate the crystallization process. An important parameter for the description of both crystal growth and nucleation is the surface free energy (surface tension), γ, of the nuclei growing on the polymer substrates. Unfortunately, in this case γ is not established unambiguously,44 but it may be used for comparisons between various substrates.

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Figure 6. Ball and stick drawings of the oxadiazole homopolymer and magnesium complexation.

Figure 7. Dependence of the rates of HAP overgrowth on oxadiazole homopolymer on 1/(ln ΩHAP)2.

The rate of nucleation, according to the “classical” nucleation theory, is45

Table 2. Surface Energy, γ, for the Overgrowth of HAP on Various Substrates



2D -βγ3u2 1 R ) 5 exp d k3T3 (ln Ω)2


In eq 6, D is the diffusion coefficient, d is the mean ionic diameter, β is the shape factor (16π/3 for the spherically shaped nuclei), u is the molecular volume of the HAP overgrowth (formula weight/9 × 6.023 × 1023 ), k is the Boltzmann’s constant, and T is the absolute temperature. In our experiments, the measured rate of crystal growth, RG, at time zero18,19 (i.e., at the end of the nucleation process) is equal to the nucleation rate R. Also in the present work, the rates of crystal growth at the time of the onset of precipitation were used for the kinetic treatment to avoid taking into account the fact that the HAP crystals formed may serve as seed crystals for further overgrowth. In a logarithmic form, eq 6 may be written as

ln RG ) ln

( )

βγ3u2 2D 1 5 3 3 d k T (ln ΩHAP)2


According to eq 7, it is expected that the rates of HAP formation vary linearly with 1/(ln ΩHAP)2. The data obtained from the kinetics of HAP overgrowth on oxadiazole homopolymers yielded the straight line shown in Figure 7. From the slope of this line, a value of 158 ( 15 mJ m-2 was estimated for the HAP overgrowth. Similar values for the overgrowth of HAP on foreign substrates are shown in Table 2. The experimental conditions in the present work were chosen so that the supersaturated solutions were stable,45,46 yet the overall free energy for the formation of the critical nucleus, ∆Gcr, will be lower than the corresponding to homogeneous nucleation ∆GN by a factor Φ, which is