Hydroxyapatite Mineralization in the Presence of Anionic Polymers

Article pubs.acs.org/crystal

Hydroxyapatite Mineralization in the Presence of Anionic Polymers Robert J. Coleman,† Kevin S. Jack,‡ Sébastien Perrier,§ and Lisbeth Grøndahl*,† †

School of Chemistry and Molecular Biosciences, and ‡Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia, Queensland 4072, Australia § Key Centre for Polymers and Colloids, The University of Sydney, Sydney, New South Wales 2006, Australia S Supporting Information *

ABSTRACT: In biological systems, hydroxyapatite (HAP) mineralization is directed by macromolecules with anionic functional domains. This study investigates the effect of four anionic polymers with varying functional groups with respect to their inhibition of HAP growth. The anionic polymers include naturally occurring alginate and heparin as well as synthesized phosphorylated alginate and poly(3-sulfopropyl acrylate) (PSPA), thus investigating the three anionic functional groups of carboxylate, phosphate, and sulfate. Crystal growth inhibition was studied through the determination of the rates of HAP crystal growth while maintaining a constant composition of the supernatant as well as by investigations of the morphology of crystals grown in the presence of the anionic polymers. It was found that neither alginate nor PSPA had a large effect on crystal growth, phosphorylated alginate showed strong nonspecific binding, and heparin displayed preferential binding to the 002 face of the crystal.

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aspartic acid and glutamic acid residues.6b Chemical modification of glutamic acid residues in BSP results in a reduction in the nucleation ability, suggesting that they play an important role in nucleation.6b The presence of phosphate groups has also been identified as necessary to nucleation by BSP7 along with DPP, due to a reduction in the nucleating ability of both molecules upon modification of the phosphate groups.6b Biomacromolecules that inhibit HAP crystal growth through surface binding (such as osteopontin) also feature domains rich in acidic functional groups.8 Therefore, different anionic macromolecules with varying functional groups apparently perform the same action in vivo, and at the same time macromolecules with the same functional groups can have very different functions. Thus, it is difficult to predict the effect of a macromolecule on crystal nucleation, growth kinetics, or morphology. The effect of polymer architecture on mineralization is a relatively new field of study, with investigations into the effect of molecular weight and block structure of model polymers on the growth rate of calcium oxalate reported.9 Additionally, the effect of model polyelectrolytes with carboxylate functionality on HAP morphology has been studied. For example, HAP crystals synthesized at 90 °C in the presence of polyaspartic acid10 or poly(acrylic acid) (PAA) in a TRIS buffer10b,11 have increased length/width ratios, as viewed by transmission electron microscopy (TEM). Furthermore, X-ray diffraction evidence showed that these reaction conditions resulted in an

INTRODUCTION The crystallization of many minerals within biological systems is modulated by biological macromolecules rich in anionic functional groups (i.e., acidic macromolecules), such as phosphoproteins and glycosaminoglycans.1 In contrast to the insoluble macromolecules that can self-organize into structural matrices, these soluble macromolecules can influence the nucleation of crystals, their growth rates, and the resultant morphology and overall size. In mammalian systems, the presence of the acidic macromolecules, bound to an insoluble matrix such as collagen, is thought to induce the nucleation of calcium phosphate phases.2 Once nucleation has occurred, the presence of anionic polymers reduces the growth rates of the developing crystals by binding to the growth sites. The adsorption of polymers to specific faces during crystal growth can lead to a decrease in the growth rate of that particular face, thus changing the final crystal morphology.3 The calcium phosphate mineral hydroxyapatite, Ca5(PO4)3OH (HAP), is the model for the mineral component of mammalian bone and dental tissue. Two distinct morphologies of HAP are observed in the form of the hexagonal prisms of dentin4 and the plate-shaped structures observed in bone.5 HAP forms within the gap regions between collagen fibrils, and, in addition, macromolecules such as bone sialoprotein (BSP), dentin phosphophoryn (DPP), and chondroitin-4-sulfate (CS4) have been implicated as initiators for the heterogeneous nucleation of HAP.6 However, the exact tertiary structures of these biomacromolecules along with the mechanism by which they nucleate and interact with HAP during crystal formation have yet to be elucidated. In BSP, there are domains rich in carboxylic acid functionalities, such as © 2013 American Chemical Society

Received: March 26, 2013 Revised: August 9, 2013 Published: August 12, 2013 4252

dx.doi.org/10.1021/cg400447e | Cryst. Growth Des. 2013, 13, 4252−4259

Crystal Growth & Design

Article

Figure 1. Chemical structures of (A) alginate (Alg), (B) phosphorylated alginate (PAlg), (C) heparin (Hep), (D) poly(3-sulfopropyl acrylate) (PSPA).

HAP of 126 mg m−2 reported in one experiment (24 °C, pH 6.8, SSAHAP 81 m2 g−1)18 and 273 mg m−2 reported in another (37 °C, pH 6.8, SSAHAP 26 m2 g−1).19 The current study investigates the effect of these anionic polymers on the rate of crystal growth using the constant composition method (which enables the maintenance of a constant level of supersaturation) and crystal morphology (evaluated from XRD and TEM) of HAP. The effects of these model polymers on the rates of HAP growth and morphology are discussed in terms of the functionality present in the molecule and their possible mode of interaction with HAP.

elongated crystal habit.10b The kinetics of HAP crystal growth have also been studied in the presence of phosphorylated pentapeptides,12 which reduced the growth rate to 20% of the uninhibited rate. The intimate association of CS4 to hydroxyapatite was determined previously through solid-state NMR,6a and thus further studies of the effect of anionic polysaccharides, in general, on the morphology and crystal growth kinetics of HAP are required. Indeed, the polysaccharide alginate (Alg, carrying carboxylate functionalities) has been reported to reduce the growth rate to 44% of the uninhibited rate.13 In order to further the understanding of the effects of different anionic groups and polymer structure on HAP mineralization, this study investigates Alg and a phosphorylated analogue of alginate (PAlg). In addition to these two sulphated macromolecules were selected: heparin (Hep) and poly(3sulfopropyl acrylate) (PSPA) which are biological and synthetic, respectively. These anionic polymers are shown in Figure 1, and they were chosen as they feature carboxylate, phosphate, and sulfate functionality and are representative of the types of functional groups that have been implicated in the biomineralization of HAP. Alg is a naturally occurring biopolymer extracted from algae or bacteria.14 Each monomer unit (mannuronic acid (M) or guluronic acid (G)) contains one carboxylate group. Alg has a high affinity for divalent ions including Ca2+ and can form physical hydrogels in the presence of such ions.15 PAlg is a synthetic derivative of Alg produced by a heterogeneous phosphorylation reaction introducing phosphate groups at C2 and/or C3.16 Our previous study demonstrated that the addition of phosphate groups to alginate caused stronger hydrogels to form due to the higher affinity of phosphate for calcium. 16 Hep is a highly sulphated glucosaminoglycan (GAG) composed predominantly of the repeating disaccharide units of iduronic acid and glucosamine as well as, to a lesser extent, the disaccharide glucuronic acid glucosamine. This results in each monomer unit containing two anionic groups (either one carboxylate and one sulfate or two sulfate groups). Hep has been reported to have a high affinity for Ca2+ with a binding constant (log K) of 4.5.17 In addition, studies have looked at heparin binding to hydroxyapatite through adsorption isotherms, with a maximum adsorption to

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EXPERIMENTAL SECTION

Reagents. Heparin sodium salt from porcine intestinal mucosa (Hep, Mw of 13 kDa, corresponding to approximately 46 monomer units)20 was purchased from Sigma Aldrich. Sodium alginate (Alg, low viscosity; 250 cps for 2% at 25 °C, sourced from Macrocystis Pyrifera) was purchased from Sigma-Aldrich and characterized to have a Mw of 141 kDa (corresponding to an average of 700 monomer units) and an M/G ratio of 1.56.16 Phosphorylated alginate (PAlg) with a degree of substitution of 0.26 was synthesized from alginate using a method previously reported and had a Mw of 39 kDa (corresponding to approximately 170 monomer units).16 Poly(3-sulfopropyl acrylate) (PSPA) with a molecular mass (Mn) of 10.8 kDa (corresponding to an average of 47 monomer units) was prepared by reversible addition− fragmentation chain-transfer (RAFT) polymerization and characterized as described in the Supporting Information. Calcium chloride dihydrate (99.0%), calcium nitrate tetrahydrate (99.0%), and potassium hydroxide (>85%) was sourced from Sigma Aldrich. Potassium dihydrogen phosphate (99%) and diammonium hydrogen phosphate (98%) were sourced from Ajax Finechem. Potassium chloride (99.5%) and ammonia 25% solution were sourced from Merck. Milli-Q water was used throughout these experiments. Hydroxyapatite Seed Crystal Synthesis. Hydroxyapatite seed crystals (sHAP) were synthesized using an adaptation of the procedure reported by Ebrahimpour et al.21 Briefly, 50 mL of 0.5 M Ca(NO3)2 was placed in a water-jacketed vessel thermostatted at 80 °C, and 25% ammonium solution was added to adjust the pH to 9. After the system was flushed with N2 gas for 30 min, 50 mL of 0.3 M (NH4)2HPO4 was added over 2 h. The resulting mixture was maintained at this temperature overnight before recovery of the precipitate by centrifugation for 5 min at 4.4 k rpm and then washing with Milli-Q water three times. The pellet was resuspended in 50 mL of water, from which 1 mL was further diluted to 50 mL, before 50 μL of this diluted 4253



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equilibrating overnight at 37 °C in 1 mL of σHAP = 0.0 solution containing Alg, PAlg, Hep, or PSPA (1 mg/mL, which is equivalent to 0.03 mg of additive/mg of sHAP). Crystal growth was initiated by the addition of the slurry to the σHAP = 3.6 solution resulting in a polymer concentration in the final solution of 10 mg/L. A TPS microchem controller and recorder with a Sentek electrode (calibrated using pH 7 and 4 buffers at 37 °C) was arranged to control two Biochemvalve diaphragm pumps in response to changes in pH. The two pumps were adjusted and calibrated to deliver 30 μL volumes of titrant. The volume of calcium titrant added was recorded as a function of time after the addition of sHAP. The rate of change of the volume of titrant addition with time (dV/dt) was calculated over 4-min intervals after addition of the sHAP. The rate of HAP crystal growth (RHAP) was calculated from each dV/dt with eq 1.25

suspension was transferred onto a holey carbon grid for TEM analysis. The remaining suspension was dried in an oven at 100 °C, and the resulting dried crystals were ground to a powder using an agate mortar and pestle. These sHAP crystals were analyzed by powder X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Xray photoelectron spectroscopy (XPS), and the Brunauer−Emmett− Teller (BET) method. Hydroxyapatite Synthesis in the Presence of Anionic Polymers. The procedure used was similar to that used for the synthesis of the sHAP crystals. The anionic polymers were added to the calcium solution at a concentration of 5 mg/L before adjusting the pH to 9. The resulting samples are labeled HAlg, HPAlg, HHep, and HPSPA for hydroxyapatite synthesized in the presence of Alg, PAlg, Hep, and PSPA, respectively. Solutions. The amounts of CaCl2·2H2O, KH2PO4, and KCl salts required to yield solutions with an ionic strength (I) of 0.15 M and solution supersaturation with respect to HAP (σHAP) of 0.00 and 3.60 were calculated by the speciation and thermodynamics program Phreeqc,22 employing the Minteq.v4 database23 and matched those published previously.24 The final calcium and phosphate concentrations are given in Table 1. All solutions were filtered through a 0.45

RHAP =

σHAP = 0.0

σHAP = 3.6

total calcium (mM) total phosphate (mM) total KCl (M) total KOH (mM) pH I (M)

0.07 0.042 0.15

0.40 0.24 0.15

7.40 0.15

7.40 0.15

titrant 1

% growth extent =

titrant 2

1.8

* mHAP × 100 mHAP

(2)

where m*HAP is the mass of HAP grown from the surface of sHAP, calculated by integrating eq 1 from t = 0 to the time point being investigated. The error in the growth rates was evaluated from leastsquares fitting of data from a single run as well as from the spread in data for six replicates. The latter was calculated to have the larger error of 8%, and this was applied throughout. Characterization. ATR-FTIR spectra were recorded on a PerkinElmer spectrum 2000 instrument coupled to a Smiths Detection single reflection diamond ATR unit. Spectra were recorded from 4000 to 550 cm−1, with a resolution of 8 cm−1 and eight repeated scans. TEM images were acquired on a JEOL 1010 instrument at 100k × magnification, with an accelerating voltage of 100 kV, a 100 μm condenser aperture, and a 20 μm objective aperture. Distributions of the length, width, and aspect ratios (length/width) were acquired for populations of a minimum of 40 individual particles across a minimum of five grid sections. The particles selected had well-defined edges and no overlap with other crystals. The aspect ratios were calculated for each particle. Imaging and size measurements were performed using the Philips Soft Imaging iTEM software package. XRD traces were recorded on a Bruker Discover instrument with Bragg−Bretano geometry using Cu Kα radiation (40 kV, 40 mA). Traces were recorded from 10 to 70° in 2θ with a step size of 0.02° and an observation time of 4 s per step. The full-width at halfmaximum (β1/2) for each diffraction peak was determined using the Reitveld refinement software Topas (Bruker AXS) with fundamental parameters (FP) peak types (determined by refining the XRD profile of LaB6). The mean persistence of order within the crystallite domain (τhkl) along the 002 axis (at 25.86° 2θ) and an axis perpendicular to this, the 310 axis (at 39.80° 2θ), were calculated using the Scherrer eq 3.

1.08 0.294 1.78 0.296

(1)

where Ceff is the effective number of moles of HAP formed per liter of titrant added (in mol L−1), mHAP is the mass of sHAP crystals, and SSAHAP is the specific surface area of the seed crystals (in m2 g−1). The % extent of growth at each of the 4-min time intervals was calculated from eq 2.

Table 1. Composition of Solutions Employed in Constant Composition and Relative Growth Rate Experimentsa solution

Ceff dV mHAPSSAHAP dt

0.003

σHAP = (αCa5αPO43αOH/Ksp)1/9 − 1; Ksp = 2.35 × 10−58; γOH− = 0.75, γCa2+ = 0.32, γPO3− = 0.08. 4 a

μm nylon filter to minimize sources for heterogeneous nucleation. The solution pH was adjusted to 7.4 using 0.15 M KOH prior to use. Minimization of carbonate ion contamination was achieved by flushing the solution with N2 gas for 30 min. The compositions of the titrant solutions used in the constant composition experiment are shown in Table 1. The concentrations of the lattice ions and supporting electrolyte in the titrant solutions were based on those previously published.24 Determination of the Relative Growth Rates of HAP. Crystal growth reactions were performed in a 3-necked water jacketed vessel containing 100 mL of a σHAP = 3.60 solution maintained at 37 °C. These crystal growth reactions were initiated by addition of sHAP, either as a dry powder or as a slurry to determine if the method of addition of the Alg had an effect on the growth rates observed. The slurry was prepared by adding sHAP to 1 mL of a σHAP = 0.00 solution which also contained 1 mg of Alg and equilibrating overnight at 37 °C. For the experiments initiated with the dry powder, the Alg was added directly to the σHAP = 3.60 solution and equilibrated before the addition of the sHAP. In both cases, a total of 30 mg of sHAP crystals was added to 100 mL of the σHAP = 3.60 solution to initiate the crystal growth. The resulting Alg concentrations in the final σHAP = 3.60 solution was either 10 or 100 mg/L (i.e., 0.03 or 0.3 mg Alg/mg sHAP, respectively) as discussed below. The resulting decrease in pH upon addition of sHAP was recorded by an Ionode IJ44 electrode connected to a Horiba D-53 m (calibrated using pH 7 and 4 buffers maintained at 37 °C). Constant Composition Growth of HAP. The experimental design of the constant composition experiment was based on that reported by Wikiel et al.12 In these experiments, the rates of crystal growth were studied while maintaining a constant composition of Ca2+ and PO43−. A 100 mL aliquot of the σHAP = 3.60 solution was purged with N2 for 30 min in a 3-necked water jacketed vessel at 37 °C. A total of 30 mg of sHAP was added to this solution, which had been

τhkl =

kλ β1/2cos θ

(3)

where λ is the wavelength, θ is the diffraction angle, β1/2 is the full width at half-maximum of the diffraction peak in radians, and k is a constant related to the crystal habit, chosen as 0.9 for a hexagonal system. The specific surface area (SSAHAP) (in m2 g−1) was determined by nitrogen adsorption/desorption on a Quantachrome quadrasorb instrument. HAP crystals were outgassed at 200 °C overnight before analysis was performed. The ratio of the nitrogen gas pressure to the saturated vapor pressure z = p/P0 ranged from 0.01 to 0.3. 4254



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Figure 2. Characterization of sHAP crystals by (A) FTIR; (B) XRD; and (C) TEM. Scale bar denotes 200 nm.

= 3.6 solution containing Alg and (ii) adding a σHAP = 0.0 slurry containing sHAP crystals and Alg which had equilibrated overnight to a σHAP = 3.6 solution. In all cases, there was an initial rapid change in pH over 2000 s followed by a more steady decrease, and the pH was monitored for a total of 15 000 s (see Supporting Information, Figure S2 for pH vs time curves). When sHAP crystals were added to the σHAP = 3.6 solution, the overall change in pH at 15 000 s (cf. that at the addition of the seed crystal) was ΔpH = −0.52. When sHAP was added as a dry powder (method (i)), the presence of Alg at a concentration of 0.03 mg of Alg/mg of sHAP yielded a value of ΔpH = −0.47, while an Alg concentration of 0.3 mg of Alg/ mg of sHAP yielded ΔpH = −0.39. These findings illustrate that Alg at these concentrations are capable of inhibiting HAP growth to some extent and that this inhibition is concentration dependent. This is in agreement with a previous study that showed a dependence of the HAP growth rate on alginate concentration.13 The alternative approach for Alg addition explored in the current study (method (ii)) was the use of a sHAP slurry in which Alg at a concentration of 0.03 mg of Alg/mg of sHAP had equilibrated overnight, resulting in a pH change ΔpH = −0.41. This is a similar growth inhibition to that of adding 0.3 mg of Alg/mg of sHAP directly to the σHAP = 3.6 solution, and this effect can be further seen by comparing the curves shown in Figure S2a−c, Supporting Information. This illustrates a clear effect of the method of Alg addition on HAP growth rates and indicates that Alg adsorbs relatively slowly to the sHAP crystal surface. This finding is in agreement with previous studies on heparin and CS4 adsorption to HAP, which require approximately 2 h to reach equilibrium.18,19 Determination of HAP Growth Rates in the Presence of Anionic Polymers. On the basis of the results obtained for the relative growth rates, a sHAP slurry containing 0.03 mg of polymer/mg of sHAP was used throughout the constant composition experiments. The amount of calcium titrant added as a function of time was recorded, and typical plots of these are shown in Figure 3. Upon the introduction of sHAP to the σHAP = 3.6 solution, there is an initial period of rapid growth akin to that of the relative growth rates experiment, before crystal growth progresses at a constant rate (i.e., a linear dependence of volume of titrant vs time). Qualitatively, it can be seen from Figure 3 that the adsorption of the anionic polymers to the surface of HAP has a significant inhibitory effect on the growth kinetics, which appears to be most pronounced when PAlg is adsorbed (Figure 3e). The volume of titrant vs time data was converted to growth rate as a function of % growth extent as described in the Experimental Section (see eqs 2 and 3), and the resulting data

XPS was performed using a Kratos Axis ULTRA X-ray photoelectron spectrometer incorporating a 165 mm hemispherical electron energy analyzer. The incident radiation was monochromatic Al Kα Xrays (1486.6 eV) at 150 W and 45 degrees to the sample surface. Photoelectron data was collected at a takeoff angle of θ = 90°. Survey (wide) scans were taken at an analyzer pass energy of 160 eV and carried out over a binding energy range of 1200 to 0 eV with 1.0 eV steps and a dwell time of 100 ms. Statistical Analysis of Crystal Sizes. Statistical analysis was performed with Statistica (StatSoft Inc.). For the measurement of particle sizes, the sets of measurements were first tested for normality by a Shapiro-Wilk W test, where a p value > 0.05 signifies that the data set forms a normal distribution. Because of some of the samples forming non-normal distributions, a Wilcoxon matched-pair test was performed to compare any observed differences in the distributions of the length, width, and aspect ratio between a HAP sample prepared in the presence of an anionic polymer and HAP prepared with no additives. For this nonparametric test, p < 0.05 implied a significant decrease in the length, width, or aspect ratio of the particle.

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RESULTS AND DISCUSSION Characterization of sHAP. The sHAP crystals used throughout the relative growth rates and constant composition experiments of the current study consisted of phase pure hydroxyapatite as evaluated from FTIR and XRD spectroscopy displayed in Figure 2, panels A and B, respectively. The FTIR spectrum shows the expected bands for phosphate and hydroxyl vibrational modes of HAP. 26 The % CO 3 2− incorporation was estimated to be 3% by the empirical relationship of Featherstone et al.,27 which uses the υ3 CO32− band at 1420 cm−1 and the υ3 PO43− band at 560 cm−1. The XRD pattern matched that of the database entry for a stoichiometric HAP with the formula Ca5(PO4)3OH and a hexagonal (P63/m) unit cell (JCPDS, Card No. 9-0432).28 From XPS, it was determined that the HAP seed crystals had a Ca/P ratio of 1.67 ± 0.17 in agreement with the chemical structure. BET measurements yielded a specific surface area of 48.8 ± 1.8 m2/g. This value falls within the range of those previously published for HAP synthesized by solution methods.12,29 The morphology of the sHAP crystals as observed by TEM and displayed in Figure 2C was primarily that of elongated structures with an average length of 91 ± 5 nm and width of 24 ± 1 nm, resulting in an aspect ratio of 3.9 ± 0.2. Effect of Alg on Relative Growth Rate. During the formation of the basic calcium phosphate salt hydroxyapatite hydronium ions (H3O+) are produced concomitantly, and therefore crystal growth can be followed simply by monitoring changes in pH with time. This approach was used to study the relative growth rates and to evaluate if there was any difference between (i) adding the sHAP crystals as a dry powder to a σHAP 4255



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3.7 × 10−8 mol min−1 m−2. The values for the growth rates lie within the standard error; however, the induction period was much greater in the current study. This can be attributed to a somewhat lower mass of HAP seed crystals used in the previous study (approximately half) coupled with a lower SSAHAP of their seed crystals (29.2 m2 g−1). While all of the polysaccharides were observed to significantly reduce the growth rate, this was not the case for PSPA (Table 2). Only minor reductions in growth rates were observed when Alg (28%) or Hep (18%) were added to the σHAP = 3.6 solution, while that of PAlg was significantly higher (62%). A previous study on HAP growth rates in the presence of alginate (where [Alg] = 2 × 10−7 mol/L at σHAP = 3.6) found a reduction from 5.4 × 10−8 mol min−1 m−2 with no additive to 2.4 × 10−8 mol min−1 m−2 with alginate present.13 This is a significantly larger reduction in growth rate (56%) than that observed in the current study for Alg (28%) where we used a concentration of approximately 7 × 10−7 mol/L. Unfortunately, the previously published study did not report the molecular weight or the M/G ratio for the alginate polymer used and it is, therefore, not possible to directly compare our results to this previous study. Particle Morphology. The morphology of HAP particles prepared in the presence of anionic polymers was observed under TEM (these images are provided in the Supporting Information, Figure S3).The observed particles were similar in shape to that of sHAP prepared in the absence of additives and displayed elongated structures with high aspect ratios. Detailed analysis of the particle dimensions are displayed in terms of histograms of the length, width, and aspect ratios with examples for sHAP and HHep shown in Figure 5 (all histograms can be found in Supporting Information, Figure S4). The mean values are listed in Table 3. On the basis of detailed statistical analysis (as outlined in the Experimental Section), all samples displayed a significant reduction in the particle length (p < 0.05), while only sample HPAlg displayed a reduction in the width. None of the samples showed a significant change in aspect ratio, although it should be noted that HHep and HPSPA displayed the lowest mean values as well as narrower distributions of the aspect ratio as illustrated for HHep in Figure 5. Crystallite Dimensions. XRD was used to evaluate the effect of added anionic polymers on the mean crystallite dimensions over which order persists along the c-axis (normal to the 002 plane) and an axis perpendicular to this (normal to the 310 plane) which were calculated using the Scherrer equation. The 002 and 310 diffractions in the XRD spectra are labeled in Figure 2B, and the dimensions over which order persists determined from the analysis are listed in Table 3. For samples HAlg and HPSPA, no significant change was observed for either dimension. For the HHep sample τ002 was reduced by 40%, while a smaller reduction (26%) was seen in the τ310 dimension (Table 3). For HPAlg, τ002 and τ310 were decreased by 22% and 35%, respectively. Effect of Anionic Polymers on HAP Morphology and Domain Lengths. TEM shows the overall shape and allows the measurements of the length and widths of the HAP particles. On the other hand, XRD was used to estimate the persistence of order in the crystallite domains that comprise the particle. Therefore, both TEM and XRD were used to evaluate the effect of anionic polymers on the morphology of HAP, i.e., on the overall dimensions of the particle and the crystalline domains within.

Figure 3. Amount of titrant ([Ca] = 1 mM) added versus time in the presence of (a) no additives; (b) PSPA; (c) Hep; (d) Alg; (e) PAlg. All anionic polymers were present at a concentration of 0.03 mg/mg of HAP.

Figure 4. Instantaneous growth rate of HAP growth as a function of % growth extent. Inset: Expansion of the region after 20% extent of growth with least-squares fit applied.

for the HAP growth in the absence of polysaccharide is shown in Figure 4 as an example. The induction time between addition of the seed crystals and establishment of a linear growth rate with time has previously been attributed to the reduction in active growth sites of the seed crystals as the environment changes from σHAP = 0.0 in the slurry to supersaturation (σHAP = 3.6).30 In all experiments, a linear growth rate was established after 20% growth extent had occurred, and the data from 20 to 24% was, therefore, used to calculate the value for the rates of HAP growth, which are tabulated in Table 2. The growth rate of HAP when no additives are present reaches an average value of 3.9 ± 0.3 × 10−8 mol min−1 m−2, which is similar to results in the literature,12 where the growth rate of HAP was determined, at the same supersaturation of σHAP = 3.6 and under constant composition conditions, to be Table 2. Growth Rates Obtained from the Constant Composition Experiments additive

none

Alg

PAlg

Hep

PSPA

growth rate (10−8 mol min−1 m−2)a reduction (%)

3.9

2.8 28

1.5 62

3.2 18

3.5 10

a

Standard error: ± 8% 4256



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Figure 5. Distributions of length, width, and aspect ratios for sHAP (a: dark gray bars) and HHep (b: light gray bars).

fully ionized at both pH 7.4 and 9; however, the phosphate group is only approximately 70% fully ionized at the lower pH. Despite these different experimental conditions, we believe it is still valid to evaluate the overall outcome of HAP mineralization from the combined data since none of the polymers induced nucleation in the σHAP = 3.6 solution (data not shown) and since it is inherently the same processes that occur in the growth phase. The decrease in HAP growth rates in the presence of alginate (10 mg/L) as well as a small reduction in particle length (5 mg/L) indicates that although some adsorption and inhibition occurs, it is likely due to weak nonspecific binding. Changes in crystal morphology upon addition of carboxyl-containing polymers have previously been studied at much higher polymer concentrations of 0.5−10 g/L.10b At these higher concentrations, a reduction in width of the particles was observed, along with a reduction in both τ002 and τ310 dimensions.10b However, a separate study investigated crystal morphology of HAP particles in the presence of PAA using a similar polymer concentration to the current study and found effects that were much smaller.34 In summary, it appears that vastly different effects can be produced by using polymer concentrations that differ by orders of magnitude. The growth rate inhibition observed in our study in the presence of Alg (140 kDa, 10 mg/ L) was relatively small (28% reduction compared to control) compared to that of PAA and PAsp; for these polymers, both concentration and molecular mass dependencies were reported, and high molecular mass polymers cause a larger reduction in growth rate.35,36 Chemical composition also plays an important role with reports of complete growth inhibition in the presence of PAsp (28.8 kDa and evaluated at 5 mg/L).35b Considering the molecular mass of Alg used in the current study, it thus appears that carboxylate-containing peptides have a larger inhibitory effect than carboxylate-containing polysaccharides on crystal growth. PAlg caused the largest decrease in HAP growth rate (by 62%) of the current study and a significant decrease in both particle and crystallite dimensions. Since the particle aspect ratio remained unchanged relative to HAP, this indicates that the introduction of phosphate functionality to Alg imparts an increased nonspecific affinity to the HAP surface when compared to Alg. This increased affinity could be due to a number of factors or perhaps a combination. First, PAlg has on average a higher number of charged groups per monomer unit than Alg, which would result in increased electrostatic interactions with HAP. Second, while the polymer molecular mass of PAlg is less than Alg, which is expected to decrease the effect on growth rate as discussed above, the two polymers do display different chain conformation,17 which could be attributed to the larger inhibitory effect observed for PAlg.

Table 3. Measurements of Crystal Dimensions by XRD and TEM XRD crystallite dimensions sample

τ002 (nm) ± 10%

τ310 (nm) ± 8%

sHAP HAlg HPalg HHep HPSPA

77.3 68.3 60.0 48.0 75.4

47.2 40.0 30.9 35.1 44.3

TEM sizes length (L) (nm)

width (W) (nm) ± 1

± ± ± ± ±

24 22 21 22 21

91 82 78 74 67

5 4 4 3 2

aspect ratio (L/W) 3.9 4.0 3.7 3.4 3.3

± ± ± ± ±

0.2 0.2 0.2 0.1 0.1

Both measurements indicate that Alg has the least effect on HAP morphology. It can be seen from Table 3 for of all the additives investigated here that the addition of Alg has the least effect on the particle length observed by TEM and no significant changes in crystallite dimensions were observed by XRD. The HPAlg sample displayed a significant reduction in both length and width of the particle by TEM as well as crystallite dimensions by XRD. However, the aspect ratio of the particles obtained by TEM was not significantly altered, and it therefore appears that, overall, PAlg adsorbs nonspecifically to the HAP surface. Both the HHep and HPSPA particles displayed reduced lengths and unchanged widths, based on TEM, and in addition they both show the smallest aspect ratios of all samples in this study. Interestingly, the aspect ratio distributions for HHep and HPSPA were narrower compared to sHAP. Of these two polymers with sulfate functionality, only the HHep sample displayed a change in crystallite dimensions based on XRD. The τ002 for HHep is significantly less (40%) than that observed for sHAP, suggesting that the long-range ordering of domains along the length of the crystals is disrupted through the binding of Hep. This indicates a strong interaction of Hep to the HAP 002 surface suggesting the occurrence of face-specific binding. Effect of Anionic Polymers on HAP Growth. The two sets of experiments (growth rates and crystal morphology) of the current study were conducted under different experimental conditions as dictated by the inherent nature of the experimental requirements: In the determination of growth rates at relatively low temperatures, low values of supersaturation are required in order to eliminate homogeneous crystal nucleation. In examining crystal morphology, nucleation and growth must both take place, and this requires much higher levels of supersaturation, with a high temperature and high pH maintained to ensure the formation of the HAP phase. Considering the pKa values for carboxylate (3.4−3.7 for Alg31 and 2.8−3.1 for Hep32), sulfate (0.5−1.5 for Hep32), and phosphate (1.5 for glucose-1-dihydrogen phosphate, 6.5 for glucose-1-hydrogen phosphate33), most functional groups are 4257



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CONCLUSION The anionic polymers of the current study display vastly different effects on the crystal growth and resultant morphology of HAP. While both Alg and PAlg showed nonspecific binding, it was found that sulfur-containing polymers, in particular Hep, displayed preferential binding to the face normal to the length of the crystal, corresponding to the 002 face. While it could be concluded that acid and phosphate containing alginate-based polysaccharides had less of an effect than peptides carrying the same functional groups, the polysaccharide Hep displayed a much greater effect than the sulfate-containing acrylate polymer of the current study. The effect of Hep on crystal morphology supports the role of other GAGs in in vivo mineralization.

Third, the phosphate and carboxylate groups have different affinities for Ca2+ in solution and would therefore be expected to also have different affinities to Ca2+ on a crystal surface. An indication of the relative affinities of carboxylate and phosphate groups for calcium can be inferred from critical stability constants for glucose-1-phosphate (log K = 2.50) and glucuronic acid (log K = 1.50) toward calcium.33 A series of highly charged pentapeptides containing carboxylic acid and phosphate groups were studied with respect to their effect on HAP growth rate.12 The PAlg experiment of the current study was run at a polymer concentration of 3 × 10−6 mol monomer/ m2 HAP, and this compares to the highest concentrations used for the pentapeptides (1−1.5 × 10−6 mol monomer/m2) where a reduction of growth rate of 80% was reported.12 Considering the significantly higher molecular mass of the PAlg polymer (39 kDa) relative to the pentapeptides, we can again conclude that based on the data available the peptides show a higher affinity for HAP than the polysaccharide. The two sulfur-containing polymers of the current study were evaluated for their effect on HAP growth rate at very similar concentrations with respect to monomer units per surface area of HAP (2.8 × 10−6 and 2.9 × 10−6 mol/m2 for Hep and PSPA, respectively). The molecular masses of the two polymers were also very similar, 13 and 10.8 kDa for Hep and PSPA, respectively. At the concentration investigated (10 mg/ L), both polymers had a limited inhibitory effect on the crystal growth rate with only Hep showing a significant reduction of 18%. In terms of morphology, both polymers resulted in a reduced particle length, with a narrowed distribution of aspect ratio, while only Hep caused a change in the mean persistence of order within the crystallite domain. It is thus clear, that for these polymers, the polysaccharide shows a larger overall inhibitory effect on HAP crystal growth than that of the polyacrylate. This can in part be attributed to the larger number of charged groups per monomer unit of Hep (two groups) than PSPA (one group) as can be seen in Figure 1. In comparison, the growth of HAP with a series of carboxymethylinulin oligomers (CMI) with different degrees of substitution (DS; number of carboxyl groups per fructose unit) clearly showed a dependence of the growth rate on concentration as well as of DS.37 For CMI oligomers with a DS of 2.5, there was a significantly larger reduction in growth rate compared to a CMI oligomer with a DS of 1.5 when using the same polymer concentration (growth rates were studied for σHAP = 9.3 at 37 °C).37 In the current study, the XRD and TEM data indicate preferential binding of Hep to the face normal to the length of the crystal, corresponding to the 002 face. Another GAG known to adsorb to HAP is CS4,19 which has been implicated in the formation of HAP in vivo, based on solid state NMR experimentation that suggested that polysaccharides, such as GAGs, are most intimately associated with bone mineral.6a The sulfate groups have been shown to be necessary to the binding of CS4 to HAP, as no adsorption occurs after desulphation.19 Similar to Hep, CS4 is composed of repeating disaccharide units, glucuronic acid and galactosamine, and like heparin, it forms a helical structure, with clusters of two sulfate groups extending from alternating sides of the helix.38 These helical structures of Hep and CS4 with sulfate clusters may result in the specific binding to the 002 face of HAP as was observed in the current study for Hep. We propose that this is one possible mechanism for the formation of the plate-shaped HAP crystals observed in mammalian bone tissue.

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis of poly(3-sulfopropyl acrylate) (PSPA) homopolymers; Figure S1: 1H-NMR spectrum of PSPA; Figure S2: Change in pH after the addition of dry seed crystal to various solutions; Figure S3: TEM images of HAlg, HPAlg, HHep, and HPSPA; Figure S4: Histograms of the distributions of length, width, and aspect ratio for sHAP, HPAlg, HAlg, HHep, and HPSPA crystals. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61 7 3365 3671. Fax: +61 7 3365 4299. E-mail: l. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility (AMMRF) at the Centre for Microscopy and Microanalysis, The University of Queensland. R.C. wishes to acknowledge The University of Queensland for funding of the project through a UQ Scholarship and an ASBTE travel award.

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REFERENCES

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Hydroxyapatite Mineralization in the Presence of Anionic Polymers

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