Direct Observation of Spiral Growth, Particle Attachment, and

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Direct Observation of Spiral Growth, Particle Attachment, and Morphology Evolution of Hydroxyapatite Meng Li, Lijun Wang, Wenjun Zhang, Christine V Putnis, and Andrew Putnis Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00637 • Publication Date (Web): 29 Jun 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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Crystal Growth & Design

Direct Observation of Spiral Growth, Particle Attachment, and Morphology Evolution of Hydroxyapatite

Meng Li,† Lijun Wang,*, † Wenjun Zhang,† Christine V. Putnis,‡,# and Andrew Putnis‡,§

†

College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China ‡

Institut für Mineralogie, University of Münster, 48149 Münster, Germany #

§

Department of Chemistry, Curtin University, Perth 6845, Australia

The Institute for Geoscience Research (TIGeR), Curtin University, Perth 6845, Australia

ABSTRACT The two main pathways for the growth of calcium phosphates are either via the addition of monomeric chemical species to existing steps or via the attachment of precursor particles. Although recent experimental evidence suggests that the particle-attachment pathway is prevalent, real-time observations for the relative contributions of monomer-by-monomer addition or attachment of particles to seed crystals remain limited. Here we present an in situ study of hydroxyapatite (HAP) (100) surface growth with long imaging times by atomic force microscopy (AFM). We observed that HAP crystallization occurred by either classical spiral growth or nonclassical particle-attachment from various supersaturated solutions at near-physiological conditions, suggesting these mechanisms do not need to be mutually exclusive. We provided, to our knowledge, the first evidence of time-resolved morphology evolution during particle attachment processes, ranging from primary spheroidal particles of different sizes to triangular and hexagonal solids formed by kinetically accessible organized 1

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assembly and aggregation. These direct observations of HAP surface growth provide mechanistic and kinetic insights into the complex biomineralization of bone and open a way for the synthesis of higher-order and morphology-controlled biomimetic materials made of precursor nanoparticles.

INTRODUCTION

Unravelling the kinetic processes of calcium phosphate (Ca-P) formation, such as hydroxyapatite (HAP, Ca10(PO4)6(OH)2), is important for our understanding of bone and tooth biomineralization,1-3 and pathological calcification in stones as well as in cardiovascular disease.4-6 The formation of HAP crystals in bone and teeth is preceded by an amorphous calcium phosphate (ACP) precursor phase,7,8 prior to the transformation to the most thermodynamically stable HAP through a multi-stage process of phase transitions.9 High-resolution cryogenic transmission electron microscopy (cryo-TEM) results have shown that HAP formation from simulated body fluid not containing any collagen which could be the substrate in bone is initiated by the aggregation of pre-nucleation clusters leading to the nucleation of ACP before the development of oriented HAP crystals.10 Indeed, HAP crystallization has been proposed to proceed through a cluster-growth model.11 Recently, a combination of in situ investigations show that these nanometre-sized clusters in Ca-P crystallization are calcium triphosphate complexes,12 and the behavior of amorphous particles may follow recently proposed precursor-based mechanisms.12 The aggregation of precursor nanoparticles and pre-nucleation clusters challenges the simple assumptions of classical crystallization theory.13,14 Classical growth mechanisms, including spiral growth and the birth-and-spread model, assume that growth units are individual molecules or ions that attach on monomolecular steps present on a crystal 2


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surface.15,16 The spiral and island growth model is the main pathway for the growth of dicalcium phosphate dihydrate (DCPD, brushite, CaHPO4·2H2O) having a higher solubility compared to HAP.17 Growth by aggregation of nanoparticle precursors and multi-ion complexes is a nonclassical crystallization pathway18 that has been widely observed in the crystallization of gypsum,19 silicalite,20 calcite,21 and even proteins,22 and these metastable amorphous nanoparticles may be directly formed on different crystal surfaces.23-25 In addition, the coexistence of classical and nonclassical pathways of crystal growth may occur simultaneously.20,26 Although recent findings have provided mechanisms of HAP crystallization from supersaturated solutions, in which pre-nucleation clusters are already present before nucleation, still little is known about the role of pre-nucleation clusters in the seeded surface growth kinetics and morphology evolution. No direct observations have been made to confirm HAP growth by classical spiral growth via the addition of monomer species or coexistence with nonclassical particle attachment, because in situ observation with long imaging times to capture relatively complete processes of HAP surface growth from supersaturated solutions is still lacking. To reveal detailed kinetics of seeded HAP crystallization, we used in situ atomic force microscopy (AFM) coupled with a fluid reaction cell to monitor the real-time (~30 hours) growth of the (100) surfaces of HAP nanocrystals at different supersaturations in the absence and presence of citrate. We showed, for the first time, the morphology evolution during HAP surface growth, in which nanoparticles with different sizes aggregated in an organized way to form triangular solids, subsequently transforming into hexagonal aggregates. Moreover, a classical spiral growth mode by the addition of monomeric chemical species (molecules or ions) was also detected by in situ AFM. Finally, we demonstrated that citrate, ubiquitously present in bone tissue, could effectively tune the sizes of Ca-P primary particles/clusters during particle attachment. These direct observations define respective 3



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pathways of monomer addition and particle attachment, providing insights into HAP crystallization kinetics and the evolution of surface structure.

EXPERIMENTAL SECTION

HAP Crystal Synthesis. The micron-sized HAP hexagonal prisms were synthesized by a molten salt method with HAP powders used as the starting material.27 The synthesized HAP crystals were separated from the solidified mass and subjected to Bruker D8X-ray diffraction (Billerica, Massachusetts, USA) to identify crystal phase(s). X-ray diffraction (XRD) pattern was collected with Bragg−Brentano diffraction geometry of 40 mA and 40 kV and 2θ range of 5-85° with a step size of 0.02° and a step time of 17.05 min-1. Growth Solutions for In Situ AFM Experiments. HAP nucleation and growth experiments in the absence and presence of citrate were made in supersaturated solutions at 25 oC. The relative supersaturation σ for HAP can be defined as =

−1=−1

(1)

where IAP is the actual ionic activity product, Ksp is its value at equilibrium (the thermodynamic solubility product for the given HAP phase, -log ( ) = 116.8 for HAP at 25 C),9 and is the supersaturation ratio. Thus, for HAP,

o

=

[( )] [( )] [( )]

(2)

where a is the activity of the Ca2+, PO43- or OH- ion. The thermodynamic database and software of SPEC 01 were used for the calculations of the activities. A range of supersaturated solutions (

!

= 8.3–18, pH =7.40, and an ionic strength (IS) of 0.15 M)

were prepared by slowly mixing of sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4) in the absence and presence of 2 µM sodium dihydrogen citrate (Sigma-Aldrich, St. 4


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Louis, Missouri), which had a negligible effect on supersaturation. Using Metrohm 876 Dosimat Plus (0.2 mL/ min), calcium chloride (CaCl2) (Fluka, St. Louis, Missouri) was added to the reaction solutions by stirring at 700 r.p.m to avoid any high local supersaturation. The solution pH of 7.4 was adjusted by the addition of sodium hydroxide (0.01 M NaOH) (Sigma-Aldrich, St. Louis, Missouri). Ultrahigh purity water (resistivity >18.2 MΩ·cm at 25 °C) was used from a triple distillation (YaR, SZ-93, Shanghai, China) and then purified with a Milli-Q system (Billerica, MA) for all solution preparations. In Situ AFM of HAP Surface Growth. In situ surface growth experiments were performed in an O-ring-sealed fluid cell using a NanoScope V-Multimode 8 AFM (Bruker, Santa Barbara, CA), operating in contact or ScanAsyst mode. The supersaturated solutions were passed through the fluid cell containing the HAP seed crystal. Crystal surfaces were imaged using commercial silicon nitride probes (Bruker, tip model DNP-S10, spring constants k = 0.35 and tip radius < 40 nm for contact mode; ScanAsyst-fluid+ tips for ScanAsyst mode, k =0.7 N/m and tip radius < 12 nm) with scan rates of 2-3 Hz. Operating in contact mode, water was passed through the fluid cell to remove the preexisting particles on the HAP surface prior to injecting the supersaturated solutions. ScanAsyst mode based on peak force tapping was used to minimize the interaction of tip and forming Ca-P clusters to decrease the deformation depths.28 This minimal force on the samples is vital for imaging initially formed nanoparticles (especially the soft amorphous particles). The corresponding height images were collected to provide quantitative height (size) information of particles formed on surfaces. AFM experiments were performed with the HAP seeds anchored by epoxy on a mica substrate, followed by continuously flowing of supersaturated solutions with syringe, which was mounted in a syringe pump (Razel Scientific Instruments Model R100-E). The experiments were conducted under constant flow, and the chosen flow rate (0.3 mL/min) was to ensure the nucleation and step growth that were independent of flow rate, i.e., 5



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surface-controlled reaction rather than diffusion control. Moreover, we compared growth on HAP and growth on mica under the same solution conditions. For ex situ imaging, the HAP seeds were placed in a beaker filled with ca. 10 mL of different supersaturated solutions with σ from 8.3 to 13.4 at room temperature for 24-48 h in order to observe nucleation and particle formation. After 24 or 48 h, the samples were removed from the solution and immediately imaged in the AFM in contact mode. Moreover, ex situ HAP dissolution experiments (10 mM sodium dihydrogen citrate at pH 6.0) were performed for 24 h. Different locations of more than three different crystals per solution condition were imaged to ensure reproducibility of the results. Scanning Electron Microscopy (SEM). AFM tip morphologies were directly investigated using ultra-high resolution field emission scanning electron microscope (FESEM, Hitachi SU8010) at an acceleration voltage of 20 kV since the Si3N4 tips have an excellent electrical conductivity. The SEM images showed that tip blunting was progressive over the course of the tip/sample interaction in time-resolved AFM images. AFM Tip Geometry Effects. Although the vertical resolution of AFM images is within sub-nanoscale, the lateral resolution depends intrinsically on the tip geometry. Tip blunting affects measurement of the width of particles and the movement distance of steps. Due to the curvature radius of AFM tips used in our experiments, as shown in Figure S1A and B, the particles in the AFM images are larger than that of actual particles. The true width of the particles can be estimated by subtracting the added width, 2b, which can be given by20 2b = 2$2(% × ℎ) − ℎ(

(3)

where % is the curvature radius of the AFM tip (40 nm of maximum tip radius as described by the manufacturer), and ℎ is the height measured by AFM. We can calculate the actual width of the particles based on Eq. 3 by measuring the height of a specific particle.20 The long-time imaging of the HAP surfaces can cause AFM tip blunting and the increase 6


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in % will translate to an increase of 2b according to Eq. 3. To estimate the change of % over time combined with existing tools, we measured the particles on the HAP (100) surface with a new AFM tip (Figure S2A and C) and compared with the same tip that was scanned in the solution at

!

= 18 for 11 h of sequential imaging (Figure S2B and D). The calibration

measurements clearly showed the increased width caused by the tip blunting, and the broadening caused by ScanAsyst-fluid+ tip can be negligible operating in ScanAsyst mode (Figure S2C, D). However, an apparent broadening was present in contact mode after 11 h of sequential imaging (Figure S2A, B). In order to calibrate AFM measurements, we collected the corresponding cross-sectional images of AFM tips by the high-resolution SEM, showing that the AFM tip radius increased from 35.8 nm (Figure S1C) to 111.1 nm (Figure S1D). Moreover, to accurately estimate the tip blunting caused by sequential imaging in contact mode, AFM tips from different imaging times were collected in order to modify the increased width 2b (Eq. 3). The temporal change in tip dimensions measured from SEM images was to estimate % and 2b as a function of imaging time. The corresponding time-dependent relationship can be rewritten by20 % = 36.6 + 7.45 × 0 2b(t) = 2223(36.6 + 7.45 × 0) × ℎ4 − ℎ(

(4) (5)

Eq. 5 was used to correct the errors (Figure S2), which showed a perfect match with error-correction of less than 3% in width in contact mode.

RESULTS AND DISCUSSION

Coexistence of Steps and Particles on the (100) Face of HAP Seed Crystals. XRD 7



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confirmed that the crystal phase of synthesized seeds was pure HAP with the absence of other Ca-P phases (Figure S3). We considered HAP as the P63/m (lattice parameters a = b = 9.43 Å, c = 6.88 Å; γ = 120°) space group29,30. With a micrometer-scale and relatively smooth (100) surface, HAP crystals can be easily imaged by in situ AFM (Figure 1A). The atomic structure of the HAP (100) face in Figure 1B shows the mirror symmetry across the [010] direction with regularly arranged calcium atoms. Individual HAP crystals exhibit hexagonal prismatic habits with six equivalent {100} surfaces31 dominated by atomically flat terraces separated by monomolecular steps with a height, that exactly matches the 0.817-nm d-spacing of the (100) face (Figure 1C-E). Moreover, we also observed preexisting particles with heights of 1.0-4.0 nm attached mainly along the [001] step edge on the (100) surfaces of seed crystals (Figure 1F, G), which may be formed by a nonclassical pathway of particle attachment.18 Spiral Growth. To better observe the advancing steps by classical spiral growth, water was first passed through the fluid cell prior to injecting supersaturated solutions, effectively removing preexisting primary particles on the HAP (100) faces (Figure 1F). Later, in situ AFM experiments were performed using supersaturated solutions at near-physiological conditions (pH 7.4, IS of 0.15 M) in contact mode. Real-time in situ AFM of the HAP (100) face during growth in supersaturated solutions revealed steps with a corresponding height of 8.21 Å, generating hexagonal hillocks by single isolated dislocations in a spiral growth pattern (Figure 2A, B), bounded by six well-defined steps (Figure 2C). Figure 2D shows the difference between raw and corrected AFM data of step advancement rates in various supersaturated solutions due to tip geometry modifications after long imaging times. According to Eq. 4, we required a more precise step growth rate, i.e., corrected data. The [001]- step velocity determined through measurement of the step advancement in successive images following the AFM tip calibration (Figure S1), was about 7 and 13 pm/s at σ

!

of

8.3 and 18, respectively (Figure 2D). The step velocity was not measurable along other 8


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directions within a measurable time frame because adjacent non-spiral steps can strongly influence the advancement of spirals by changing the angle between adjacent steps of spirals. These in situ observations are consistent with classical predictions.13,15 Crystal growth near equilibrium is commonly described by the terrace-ledge-kink (TLK) model with a typical spiral growth pattern.32,33 The [001]_ step velocity shows a linear dependence on supersaturation (Figure 2D). According to the classical surface growth mechanisms,34 the addition of a solute molecule into a crystal structure involves a series of kinetic processes such as dehydration, adsorption, diffusion, incorporation of growth units into the crystal structure and removal of the latent heat of crystallization.35 The incorporation of growth units into the crystal structure is rate-limiting, which can be estimated by the step kinetic coefficient β by 7 = β89 Ω

(6)

where ; is the movement velocity of a straight step, Ω (2.64 × 10-22 cm3) is the molecular volume in the HAP crystal, 89 (14.85×1014 molecule/cm3 ) is the equilibrium concentration.36 The kinetic coefficient is a measure of the kinetics of adsorption, diffusion and incorporation, and should be proportional to kink-site density, n, and the net activation energy for attachment, E, through: β ~ >exp (−B/DE)

(7)

where D is Boltzmann’s constant and E is the absolute temperature. Using the corrected data by the tip calibration in Figure 2D, we calculated the step kinetic coefficient β for the [001]_ steps to be 1.50 × 10-4 cm/s at room temperature, which is much smaller than that of most inorganic salts (about few to 10-3 cm/s),37,38 but similar to those of protein and virus crystals (10-4 to 10-5 cm/s).39,40 β of 0.41 × 10-4 cm/s was also reported by Onuma et al. during the HAP island growth.36 As a consequence, the potential sources for relative low values of β in HAP spiral growth include a large activation energy for attachment after dehydration of 9



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monomeric species (ions or molecules), low surface diffusivity and difficulty for ions to incorporate into the crystal structure along a step orientation. This is consistent with the observations that steps can hardly move from a new spiral even at a relatively long reaction time (almost 10 hours) at = 13.4 (Figure S4). Indeed, incorporation of ions or molecules into the crystal structure must overcome a large energy barrier during HAP surface growth. Moreover, the HAP solubility is the most important parameter determining the rates of monomer-by-monomer addition. As the solubility drops from molar to submicromolar levels, at the same supersaturation the rates of monomer addition drop by a factor of about 1010.18, 41 Therefore, a particle-mediated HAP growth pathway may be energetically favorable and therefore may dominate during crystallization. Particle Attachment and Morphology Evolution by Organized Aggregation. By changing the scanning mode from contact to ScanAsyst in order to minimize the potential dislodgement/removal of particles caused by the movement of the AFM tip, we were able to image in situ the formation of a uniform population of particles with heights of 3.0-4.0 nm on the HAP (100) surface (Figure 3A), and they were present for a long time. The long-term existence of these particles strongly suggests that they were thermodynamically stable. The number density of particles increased, but their sizes were unchanged with reaction time from 45 to 265 min (Figures 3B-E and B’-E’). After 265 min of reaction, the particles (3.0-4.0 nm in height) coexisted on the surface with triangular- or hexagonal-shaped organized aggregates (Figures 3E) retaining an average height of 3.0-4.0 nm, width of 90-110 nm, and length of 110-120 nm (Figure 3E’, I, J). After 700 min at the late stage, the particles eventually merged to become isolated aggregates with triangular and hexagonal shapes (130-150 nm in width and 160-200 nm in length) (Figure 3F). For comparison, on a mica substrate these particles (3.0-4.0 nm in height) were also present, but organized aggregates were absent (Figure S5). In addition to these organized aggregates, some larger irregular precipitates underwent 10


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three-dimensional (3D) growth (Figure 3F-H). Measuring their height contours, such as line scan l5-6 (Figure 3F, G) at different time points, showed an increase of 1.3-2.2 nm in height along the [100] direction (Figure 3K). All the organized precipitates are aligned or 'point' in the same direction/orientation in Figure 3F-H, suggesting that the origin of such long-range alignment is most likely related to epitaxial growth, that would be energetically expected for oriented attachment at preferential sites on the HAP (100) surface. Additionally, the presence of forces between interfacial solution and solid/particle structure drives particle motion,18 this may induce particles to align in the same direction.18 Furthermore, we also noted that the orientation of triangular particles appears to be different than that in observations made during HAP dissolution experiments.42 The most likely explanation is the difference between triangular particles formed by aggregation-based growth and triangular etch pits formed by dissolution. The formation of particles on the surface initially is common for all crystal materials as high supersaturation is experienced on the crystal surface in the last moments of dehydration after retrieval from a supersaturated solution, and it is not clear how the AFM data distinguish between 2D nucleation and pre-nucleation cluster formation. Thus, we further imaged the (100) face at different time points, and observed the presence of smaller particles (about 0.9-1.0 nm) (Figure S6) with sizes resembling those of particle precursors, in agreement with our previous observations of Posner’s clusters.43 Computer representation of the Posner’s cluster with a composition of Ca9(PO4)6 demonstrates a diameter of ~0.95 nm.10 Moreover, the high-resolution cryo-TEM images of the pre-nucleation species gave 1.3 ± 0.3 nm for the diameter and 0.9 ± 0.3 nm for the height of the complexes.12 These complexes aggregated and took up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca2(HPO4)32- clusters.12 The number density of precursors of 1-nm particles decreased in favor of a growing population of larger particles (3-4 nm), i.e., by a direct aggregation of 11



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smaller particles resulted in the formation of larger particles (3-4 nm) that must be energetically favorable. These above results may suggest a growth mechanism of nanoparticle attachment. In order to confirm this, we collected continuous AFM images and observed that initial precipitate size (about 1.0 nm) was the same as precursor sizes measured as the average height (Figures S6 and 3I). Moreover, an obvious increase in the particle width was observed (Figure 3K), reflecting growth via particle attachment rather than monomer addition in the directions (Figures 3H, L, and S7). This is because the rate of monomer addition is too slow to be observed during the reaction time. Furthermore, surface relaxation processes exhibited, including post-attachment fusion, rearrangement or dissolution of precursors (about 1.0 nm) or their aggregates (3.0-4.0 nm) (Figure S8). At the subsequent growth stages, particle attachment through the organized aggregation of primary spheroidal particles, results in two-dimensional growth of hexagonal solids with an average height of 3-4 nm (Figure 4A-C). Moreover, some parts of the particles show a deviation from a hexagonal form presenting a somewhat rounded morphology, approaching an elliptical or spear head form.This could be an indication of a strong anisotropy within the system (Figure 4A-C). Some of the hexagonal precipitates tended to form stacks, whose thickness also increased several times by three-dimensional growth (Figure 4D). The height of 3-4 nm is in excellent agreement with the thickness of HAP nanocrystals and packed HAP nanoplatelets in bone.45-47 The consistency of thickness indicates characteristic of HAP crystallization by the attachment and fusion of particles with an average height (thickness) of 3-4 nm. Consistent with the morphology of these hexagonal aggregates, AFM dissolution reactions of HAP seed crystals also demonstrated that the hexagons evolved into triangular etch pits (Figure 5), in agreement with the observation of the change of pit shape.42 The etch pit shape evolution during dissolution also provides fundamental insights into growth leading to defect structures, 12


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accounting for the observed shape change of triangular pits and the organized hexagonal solids built by primary spheroidal particles. This can be further rationalized that HAP dissolves most likely by the removal of the same structural units (particles with 3.0-4.0 nm in height) with similar particle-attachment-detachment mechanisms. Role of Citrate In Particle Attachment. In the presence of 2 µM citrate in the same supersaturated solution (σ

!

= 13.4) as the experimental conditions used in Figure 3, we

further observed that the stability of the Ca–P nanoparticles/nanoclusters in heights of 1.2 ± 0.3 nm, formed at the earliest stages, was enhanced by limiting their aggregation, and larger particles (about 3.0-4.0 nm) were hardly detected (Figures 6A-C, B’, C’ and S9, 10). The number of these smaller nanoparticles increased with time until almost complete coverage of the HAP seed surface occurred (Figure S10D). The stabilization of 1-nm particles in the presence of citrate may point toward the mechanism of a kinetic stabilization. We noted that some complex features in our observations are most likely related to the influence of citrate molecules on smaller particles (about 1 nm) (Figure S10). Meanwhile, we observed an obviously preferred alignment parallel to the [001] direction (Figure S10), which is different from the particles formed on HAP in the absence of citrate (Figure 3B-D). This indicates a dual role of citrate in HAP growth: stabilizing smaller Ca-P nanoclusters and modulating an oriented adsorption along the [001] direction. Simulation results have shown that citrate can bind strongly with the calcium ions along the c-axis on the HAP (100) surface,48 and is capable of regulating bone crystal growth by preferential adsorption in specific directions. Thus, citrate in supersaturated Ca-P solutions may also adsorb to the HAP seed surface to induce Ca-P nanoclusters aligned along the calcium-rich [001] direction. Mineralized bone tissues show a complex hierarchical structure with highly organized embedded nanometric building blocks.49,50 The currently accepted model of bone mineral is ∼50- to 150-nm thick stacks of very closely packed apatitic platelets, each of the order 2.5–4 13



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nm in thickness, arranged so that their large (100) faces are parallel to each other and their c axes are strongly ordered (parallel to collagen fibrils controlling the HAP growth).51-53 Without precise regulation and control, such as matrix templates and organic macromolecules,54-59 pathological mineralization can easily occur, resulting in fragile bones.60,61 Citrate as a small organic molecule is also abundant in bone and can effectively control the intermediate phases of crystallization and the final morphology of HAP.62 The strongly bound citrate in bone covers apatite at a density of about 1 molecule per 4 nm2 (ca.1/6 of the available surface area) and accounts for 5.5 wt% of the organic matter in bone.45 Almost 80 wt% of the total citrate is associated with non-stoichiometric and plate-like HAP.63 Carboxyl groups of citrate can drive ACP directly into HAP crystals and control the size of nanocrystals by specific adsorption to the (100) surface.64,65 According to our observations, the presence of smaller nanoclusters (about 1.0 nm) stabilized by citrate can act as “cement” to fill the pores/voids among larger particles to reduce porosity. Indeed, these smaller clusters adsorbed by citrate can effectively mediate HAP surface growth by rapid and oriented adsorption along the c axis of the HAP surface to reduce defects existing among particles.65-67 This will play an important role in controlling HAP growth in bone. Recently, a combination of solid-state NMR spectroscopy, X-ray diffraction, and first principles calculations showed that citrate anions reside in a hydrated layer of a double salt octacalcium phosphate (Ca8H2(PO4)6·5H2O, OCP) citrate, bridging between apatitic layers.68 Such a structure of OCP-citrate-like hydrated layers can explain a number of known structural features of bone mineral.68 These collective results further suggest, in addition to the role of collagen in controlling HAP growth, pure HAP surface growth in the absence and presence of small additive molecules are also relevant. These HAP surface growth features in the absence and presence of citrate demonstrate a particle-based growth model that is consistent with previous observations of growth of the 14


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HAP c-face.69 The earliest Ca-P particles exhibited typical sizes of 1.0 nm and 3.0-4.0 nm in height with a narrow size distribution (Figures 3 and 6), that were also identical to the diameter of nanoparticles measured by cryo-TEM.70 In contrast to classical crystal growth, crystallization by particle attachment occurs by the addition of higher-order species ranging from multi-ion complexes to amorphous nanoparticles to nanocrystals.18 In the present study, the primary particles/clusters formed at the early stages of Ca-P crystallization in supersaturated solutions (σ

!

= 8.3-13.4) are about 1.0 nm in height, and they can then

grow by exchanging monomers with other particles, as well as through collision and coalescence events to generate particles with particular sizes of 3.0-4.0 nm in height (Figures 3B-D, S9). The formation of 3.0-4.0 nm particles becomes thermodynamically favored/stabilized possibly by the entropy and can be present for over 7 hours (Figure S8). These particles, through the association of Ca-P complexes and clusters,10,12 aggregate to form larger organized hexagonal solids (Figure 4). A recent result indicated that the aggregation of nonequilibrium clusters forms a calcium-deficient amorphous phase I [Ca(HPO4)1+x·nH2O]2x−) early in the induction period, which slowly transforms to amorphous phase II [Ca(HPO4)·mH2O] by dehydration.71 By means of synchrotron X-ray absorption near-edge spectroscopy at the calcium K-edge, the most abundant clusters were detected to be Ca(η2-PO43−)2L2 (L = H2O or η1-PO43−) in solution at the initial stage of crystallization.72 Consequently, the solution may contain a distribution of monomers, complexes, and clusters, all of which may contribute to nucleation and growth.18 Following nucleation, the newly formed phases grow through many competing processes dominated by particle attachment or monomer addition, depending on numerous factors associated with the free-energy landscape of

the

growing

crystal

surface

and

the

kinetics.18

Crystal

growth

rates

by

monomer-by-monomer addition roughly scale with solubility73 and the solubility of HAP is very low, the rates of monomer addition are thus also very low (Figure 2). In fact, the growth 15



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of individual HAP particles is limited because the growth rate is much smaller than the nucleation rate. This can be explained because a Gibbs free energy ∆G=-RTln(IAP/Ksp), to transfer an average ion from a supersaturated solution to a theoretical particle is far less than zero, consistent with observation that the formation of nanoparticles at the crystal surfaces of HAP or in supersaturated solutions, that were used in this study, is spontaneous.74 Moreover, due to the presence of the competition between monomer-by-monomer growth and growth by attachment of particles of different sizes (1.0 nm or 3-4 nm) and surface relaxation, both attached and dispersed particles can grow or dissolve (Figure S8), depending on their radii of curvature18. In fact, smaller particles (about 1.0 nm) attach rapidly to effectively smooth the interface (Figure 3K) and fill pores between particles (Figure S8). This may suggest particle attachment on HAP and subsequent “smoothing” to form a flat face. Because initial nucleation from supersaturated solutions generates a polydisperse population of nanoparticles, their assembly typically leads to an irregular morphology of solids.18 However, in the present study, one of the potential roles of particle attachment is to guide the resulting structures of HAP that exhibit unexpected morphologies from triangles to hexagons during the kinetic processes of particle attachment (Figure 3). These discrete nanoparticles as building blocks, with controlled chemical composition and size distribution, are readily formed on seeded crystals, but their assembly into well-defined morphology and complex structural organizations amenable to the natural morphology of bone nanocrystals remains difficult to understand. The self-assembly of structures with precisely defined morphologies is inherently complicated, requiring the initial formation of spheroidal nanoparticles, followed by further processing such as template-patterning75 or interfacial modification.76 Such organized solids can retain features of pores and internal microstructures formed during growth. These pores as potential internal defects may influence the etch pit morphology (Figure 5), although external morphology alone does not prove formation by a 16


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particle-based growth process because coarsening or recrystallization can subsequently obliterate characteristic signatures.18,77,78 However, our direct AFM imaging throughout HAP crystallization at different time points can detail the kinetics of growth via the particle attachment mechanism (Figure 3). The actual phase of theses particles/clusters formed in a supersaturated solution or on HAP surfaces is still not known, based only on AFM observation, but it has been shown that the precipitation of Ca−P phases may occur through the initial formation of various precursor phases, including ACP, brushite (DCPD)/monetite (DCPA),79 and OCP, prior to the transformation to the most thermodynamically stable HAP.9

CONCLUSIONS

In this study, the growth dynamics and morphology evolution of HAP crystals have been observed in situ in real time at the nanometer scale using AFM. We demonstrated that HAP growth occurs via the addition of both Ca-P monomeric species and precursor particles, bridging classical and nonclassical mechanisms (Figure 7), i.e., HAP crystals grow by the attachment of different size particles (~1 nm or 3-4 nm in height) to form organized assemblies/aggregates with triangular and hexagonal morphologies, as well as the addition of monomeric species at step edges during spiral growth. Prior to the formation of a new phase and final bulk HAP crystals, the monomers of ions and molecules can form complexes or aggregate into clusters and nanoparticles. Relative contributions of the addition of nanoparticles are much larger than that of monomer addition during HAP growth. For the first time, individual monomer additions by spiral growth on the (100) face of HAP crystals have been resolved, and step advancement rates were used to determinate the kinetic coefficient β. In addition, for a nonclassical pathway, HAP crystallization from supersaturated solutions involves initially formed amorphous spheroidal aggregates with different sizes including 1.0 17



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nm and 3.0-4.0 nm that evolve into triangular and hexagonal solids. This is brought about by a novel mechanism involving complex organization and interfacial interactions that occur simultaneously, although the final amorphous-to-HAP transformation is unknown. In situ dissolution revealed mechanisms for defect incorporation during organized aggregation of primary particles, controlling the growth rate and morphology evolution. These observations of competing pathways for HAP growth will improve fundamental understanding of natural processes such as biomineralization of bone. Finally, we provide direct evidence that citrate can play a dual role in stabilizing Ca-P clusters and inducing oriented adsorption of nanoclusters and nanoparticles on the HAP surfaces. The detailed surface growth kinetics of HAP may be further extendable to other Ca-P materials, such as OCP, and facilitate the potential design of biomimetic bone-like materials with desired functional properties, such as strength and compatibility, for use in medical bone reconstruction surgery. ASSOCIATED CONTENT

Supporting Information. Calibration of AFM tips (Figures S1, S2); XRD of synthesized HAP crystals (Figure S3); AFM of spiral growth of HAP at = 13.4 (Figure S4); AFM of ex situ formation of particles on mica (Figure S5); AFM images of particles of different sizes formed during HAP growth under different reaction conditions (Figures S6-S10); This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding author *Phone: +86-27-87288382; e-mail: [email protected] 18


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (41471245, 41071208), a Specialized Research Fund for the Doctoral Program of Higher Education (20130146110030), and the Fundamental Research Funds for the Central Universities (2662015PY206). C.V.P. and A.P. acknowledge the receipt of the European Union Marie Curie initial training networks CO2 React, FlowTrans and Minsc.

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Figure 1. Seed crystals of synthesized HAP hexagonal prisms. (A) AFM defection image of six equivalent HAP (100) faces elongated along the [001] direction. (B) The atomic structure of HAP (100) faces with a mirror symmetric axis across the [010] direction (Ca, green; P, pink; O, red; H, white) (C) AFM image of the monomolecular straight-edged and non-straight steps on an HAP (100) surface showing (D) the step height of 8.17 ± 0.28 Å (n = 100) along line l1-2 in (C), corresponding to the interlayer distance between (100) surfaces (d(100) ≈ 0.817 nm). (E) The step height is 8.17 Å as marked. In addition to the relatively smooth HAP (100) surfaces, HAP surfaces are also covered by (F) preexisting particles on step edges and terraces. (G) The average height of these preexisting particles is about 1.0-4.0 nm measured by the height profile of l3-4 in (F).

29



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Figure 2. Imaging spiral growth and quantitative determination of HAP growth rates. (A, B) Hexagonal growth hillocks on the HAP (100) surface formed by a single dislocation at σ

!

= 18. The step height of 8.21 Å is highlighted by a white dotted line in (A). Arrows show the presence of initially formed particles in (A) and their removal through the tip scanning in contact mode in (B). (C) Six unique growth steps and segment orientations for a single molecular layers are shown by the (+) and (–), and the critical length is defined by Lc. (D) Step velocity along the [001]- direction as a function of different supersaturations (σ

!

=

8.3-18) showing linear kinetics. The black and red lines are the linear fit with the raw data and corrected data by the AFM tip calibration (R2 =0.89), respectively.

30


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Figure 3. The growth of HAP crystals by particle attachment and morphology evolution on the HAP (100) surface. AFM time sequence (height images in ScanAsyst mode) showing: (A-D) in situ nucleation kinetics of Ca-P particles and an increase in total number of particles with reaction time from 45 to 210 min; (E-H) the growth shape evolution during about 30 h of continuous scanning at room temperature from a supersaturated solution at σ ! =13.4 (pH 7.4, IS=0.15 M). The red lines in (A-E) provide a reference point of a step. (B’-E’) Height profile of the initially formed particles showing that the average height of Ca-P particles is about 3.0-4.0 nm, that remained almost unchanged after 210 min of reaction time. After 265 min, the organized aggregation of the nucleated particles led to the formation of quasi-trigonal 31



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particles with an angle of 80-82° and hexagonal particles with an angle of 85-92° shown by arrows in (E, F). (I, J) Height measurement of the developing particles along lines l1-2 and l3-4 in (E) (about 3-4 nm). (F, G) Following a long period of reaction time (700 min), the number of triangle-shaped and hexagonal particles increased. (K) Height profiles of line l5-6 in (F) and (G) at a time interval of 120 min indicate solid growth by attaching particles (about 1.3-2.2 nm in height). (L) After 29.5 hours of reaction time, larger particles formed (about 120.1 nm in height along line l7-8 in (H)) as a result of 3D nanoparticle attachment.The alignment of the growth particles on the HAP surface indicates a probable epitaxial control exerted by the substrate.

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Figure 4. (A) Ex situ AFM image of larger hexagonal particles (shown by a dotted hexagon and an arrow) on the same HAP substrate as Figure 3, forming from a supersaturated solution at σ ! =13.4 for 2 days at room temperature. (B, C) The average thickness (about 3-4 nm in height, Hs shown in A) and angles of hexagonal forms (n = 50 for measured hexagonal solids). (D) The cross-sectional analysis showing some hexagonal forms can stack to form 3-D structures as shown by line l1-2 in (A).

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Figure 5. Morphology evolution of etch pits on the HAP (100) surface. (A) AFM height image showing the formation of asymmetric hexagonal etch pits in 10 mM citrate solutions at pH 6.0 for 24 hours. (B) Schematic angles of hexagonal etch pits are the mean values ± standard deviation from 20 measurements. (C) AFM deflection and (D) height image showing a hexagon-like pit. (E) Schematic illustration of etch pit shape evolution from hexagons to triangles. Arrows indicate schematically the relative step retraction rates. (F, G) Depths of etch pits along lines l1-2 and l3-4 in (C) showing the presence of both molecular steps (about 0.77-0.9 nm) and macrosteps with 3.3 nm and 9.7 nm.

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Figure 6. Modulation of Ca-P nucleation by citrate. (A-D) A sequence of in situ AFM height images of the formation of Ca-P particles at σ

!

= 13.4 in the presence of 2 µM citrate. (B’,

C’) The corresponding height distribution of nanparticles formed in (B, C) showing smaller nanoparticles/nanoclusters with heights of 1.2 ± 0.3 nm.

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Figure 7. A schematic illustration of combined classical and nonclassical pathways for HAP crystallization. HAP crystals grow by the attachment of different size particles (~1 nm or 3-4 nm in height) to form organized assemblies/aggregates with triangular and hexagonal morphologies, and the addition of monomeric species at step edges during spiral growth. Prior to the formation of a new phase and final bulk HAP crystals, the monomers of ions and molecules can form complexes or aggregate into clusters and nanoparticles. These high-order species of nanoparticles and trianglular/hexagonal solids may be amorphous.

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For Table of Contents Use Only

Direct Observation of Spiral Growth, Particle Attachment, and Morphology Evolution of Hydroxyapatite

Meng Li,† Lijun Wang,*, † Wenjun Zhang,† Christine V. Putnis,‡,# and Andrew Putnis‡,§

TOC graphic Synopsis In situ AFM approach to study hydroxyapatite surface crystallization via the addition of both monomeric species and precursor particles, bridges classical and nonclassical mechanisms. These direct observations of both spiral growth and particle attachment provide mechanistic and kinetic insights into the complex biomineralization of bone and tooth.

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Direct Observation of Spiral Growth, Particle Attachment, and

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