In Situ Atomic Force Microscopy Imaging of Octacalcium Phosphate

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In Situ AFM Imaging of Octacalcium Phosphate Crystallization and Its Modulation by Amelogenin’s C-Terminus Shanshan Wu, Menghan Yu, Meng Li, Lijun Wang, Christine V Putnis, and Andrew Putnis Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00129 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 23, 2017

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Crystal Growth & Design

In Situ AFM Imaging of Octacalcium Phosphate Crystallization and Its Modulation by Amelogenin’s C-Terminus

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Shanshan Wu,† Menghan Yu,† Meng Li,† Lijun Wang,*, † Christine V. Putnis,‡,§ and

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Andrew Putnis‡,¶

5 6 †

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College of Resources and Environment, Huazhong Agricultural University, Wuhan

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430070, China ‡

9 10 11

§

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

Department of Chemistry, Curtin University, Perth, Western Australia 6845, Australia ¶

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

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Australia 6102, Australia

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Corresponding author

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*College of Resources and Environment, Huazhong Agricultural University, 1 Shizishan St.,

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Wuhan 430070, China. Phone/Fax: +86-27-87288382. E-mail: [email protected]. 

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ACS Paragon Plus Environment


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ABSTRACT

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Amelogenin proteins play a critical role in controlling crystal growth and orientation into the

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highly organized calcium phosphate (Ca-P) minerals during tooth enamel formation. However,

29

real-time observations for understanding the kinetics and mechanisms of Ca-P surface

30

crystallization and its modulation by amelogenin have been lacking. We monitor the kinetics of

31

the (100) surface growth of octacalcium phosphate (OCP) with precisely defined

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thermodynamic driving forces in the presence of amelogenin’s C-terminus peptides inside a

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fluid cell of an atomic force microscope (AFM) with a controlled near-physiological

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environment. During in situ growth via a nonclassical particle attachment pathway, an obviously

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elongated aggregation of Ca-P nanoparticles induced by the assembly of amelogenin’s C-termini

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was observed. The nanostructured fibrous assemblies, reminiscent of extracellular matrix, are

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able to bind Ca-P nanoparticles and direct OCP mineralization. This was analyzed and

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rationalized through single-molecule determination of the binding free energy of the C-terminal

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fragment adsorbed to the (100) face of OCP. Combining in situ growth kinetics with force

40

spectroscopy reveals the shape evolution from spherical particles to elongated nanorods

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resembling the nanostructure of enamel crystallites. The findings improve the fundamental

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understanding of natural biomineralization through nonclassical crystallization routes and

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amelogenin self-assembly.

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INTRODUCTION

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Tooth enamel, as a highly organized mineralized tissue, is the result of interactions between

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matrix proteins and calcium phosphate (Ca-P) mineral surfaces, and its formation occurs in the

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extracellular matrix of a growing tooth through complex cellular and molecular events,1 such as 2


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secretion2 and self-assembly3 of matrix proteins, protein-mineral interactions,4 and proteolysis.5-7.

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Amelogenin (Amel) constitutes more than 90% of the total proteins found in the matrix in

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developing enamel.1,8 Much evidence shows that Amel proteins self-assemble into nanospheres

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and nanochains, playing a potential role in guiding nucleation and growth of Ca-P crystals.9,10

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Highly conserved full-length Amel is mainly composed of hydrophobic residues and a relatively

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shorter hydrophilic C-terminus.11,12 This charged C-terminus has significant Ca-P-binding

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affinity,13-16 suggesting that it is able to interact with certain crystal surfaces, thereby controlling

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the morphology of growing enamel crystals. Iijima et al. have shown that the Amel’s C-termini

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can

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Ca8(HPO4)2(PO4)·5H2O),17 causing apparent “elongation” and “thickening” of the crystals.18 A

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combination of the single-molecule force and molecular simulations has demonstrated that the

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C-terminal fragment exhibits a higher binding ability to the (100) face compared to the (001)

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face of hydroxyapatite (HAP, Ca10(PO4)6(OH)2), accounting for the c-axial elongated growth of

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enamel crystals.13

dramatically

change

the

crystal

shape

of

octacalcium

phosphate

(OCP,

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At the early stages of the enamel formation, initial enamel crystals were detected

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separate from the adjacent dentine, and electron-microprobe analyses revealed that early

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enamel crystals were OCP or tricalcium phosphate.19 Supramolecular aggregates of Amel and

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enamelin provide the microenvironment for the nucleation and crystal growth19 through the

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protein–mineral interactions that are a crucial factor underlying the hierarchical structure of

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the organized and elongated ribbons. Previous results have shown that the multi-steps involve

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the formation of HAP from amorphous calcium phosphate (ACP) to OCP to the final product

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of HAP.20-23 As an intermediate metastable phase, OCP structurally resembles HAP24,25

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because HAP epitaxially grows on the (100) face of OCP.26-28 An in situ dissolution study of

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the (100) face of OCP revealed a possibility of the phase transformation from OCP to HAP by

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a pseudomorphic transformation,29 providing a direct clue about the HAP formation via the 3



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OCP intermediate phase.

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Although extensive methods to probe these processes are well established for matrix

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proteins and their control over Ca-P crystallization, real-time observations for understanding

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the kinetics and mechanisms of in situ OCP surface crystallization and its modulation by

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Amel have been lacking. The aim of the present study is to explore the role of Amel’s

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C-termini in controlling surface growth of OCP, a precursor phase of enamel crystals.24 In this

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context, we hypothesize, that such a role is important, but that the nature of assembled or

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disassembled Amel’s C-termini will strongly influence its significance. To test this hypothesis,

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a series of in situ AFM experiments combined with the single-molecule force determination

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were performed, in which an OCP (100) surface interacted with different concentrations of

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Amel’s C-terminus peptides in various supersaturated solutions with respect to OCP under

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mimetic-physiological conditions. We demonstrated that a nonclassical crystallization

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pathway was exhibited for the OCP surface growth, and a clearly elongated aggregation of

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Ca-P nanoparticles induced by the assemblies of Amel’s C-termini was observed, i.e., the

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crystallographic c axes of OCP were aligned with the long axes of the peptide assemblies.

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However, relatively high concentrations of Amel’s C-terminus peptides had no effect due to

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the disassembly of peptide oligomers/particles induced by the OCP crystal surfaces.

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EXPERIMENTAL SECTION

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Synthesis and Fluorescence Labeling of 13-Mer Amel’s C-Terminal Peptides. The

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C-terminus peptide fragments (Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp) of

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Amel were synthesized according to standard procedures of solid phase peptide synthesis (GL

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Biotechem, Shanghai, China) and were purified by C18 reversed-phase high-performance

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liquid chromatography (HPLC).14,30 Moreover, the 13-mer C-terminus peptides containing 4


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fluorescein

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(Trp-Pro-Ala-Thr-Asp-Lys-Thr-Lys-Arg-Glu-Glu-Val-Asp-(FITC)-NH2) were synthesized

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and cleaved by 5 mL of TFA/thioanisole/ethandithiol/anisole (90/5/3/2) and were purified by

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C18 reversed-phase HPLC.31

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OCP Crystal Synthesis. OCP crystals were synthesized by previously reported methods,29,32

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and were characterized as single phase by X-ray diffraction (Bruker D8, Billerica,

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Massachusetts) (Figure S1). Rietveld refinement was performed using the structural model of

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OCP (JCPDS, PDF# 44-077833, 34). Synthetic OCP crystals as seed substrates were used for in

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situ AFM surface growth experiments.

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Supersaturated Solutions for OCP Surface Growth. OCP surface growth experiments in

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the absence and presence of Amel’s C-terminus peptides were made in supersaturated

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solutions at 25 °C. The relative supersaturation σ for OCP can be defined as

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isothiocyanate

𝜎=

#$% &'(

−1=𝑆−1

(FITC)

(1)

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where IAP is the actual ionic activity product, Ksp is its value at equilibrium (the

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thermodynamic solubility product for the given OCP phase,35 -log (𝐾-. ) = 96.6 for OCP at 25

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°C) and 𝑆 is the supersaturation ratio. The thermodynamic database and software of SPEC 01

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were used for the calculations of the activities. A range of supersaturated solutions (𝜎/01 =

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1.77–1.98, pH = 6.50, and an ionic strength (IS) = 0.15 M) were prepared by slowly mixing of

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sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, Missouri), calcium chloride (CaCl2)

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(Fluka, St. Louis, Missouri) and potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich,

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St. Louis, Missouri). The Amel’s C-terminus peptide stock solutions were added to each

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supersaturated solution to make the peptide concentrations at 1, 50, or 100 nM prior to pH

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adjustment. The peptide stock solution was prepared by dissolving 1 mg lyophilized peptides

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in 100 mL water and then was diluted into 10 mM Tris-HCl buffer. The pH value was finally

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adjusted to 6.5 with 0.8 mol L−1 KOH solution using Metrohm 888 Dosimat Plus (Herisau, 5



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All

supersaturated

solutions

were

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Switzerland).

prepared

using

pure

water

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(resistivity >18MΩ-cm at 25 °C, pH 5.8-6.0) from a two-step purification treatment including

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triple distillation (YaR, SZ-93, Shanghai, China) and deionization (Milli-Q, Billerica, MA,

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USA). The experimental conditions are summarized in Table S1.

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Imaging OCP Surface Growth by In Situ Atomic Force Microscopy (AFM). All in situ

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OCP surface growth experiments were performed using a Bruker MultiMode VIII AFM

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(Santa Barbara, CA) operating either in contact mode or ScanAsyst mode. An optically clear

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OCP crystal was cleaved to expose a fresh (100) surface. The supersaturated (σOCP=1.77, 1.86

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or 1.98, IS = 0.15 mol L-1, pH 6.5) solutions in the absence and presence of Amel peptides

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were passed over the cleaved OCP crystals inside the AFM fluid cell at a constant flow rate of

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0.5-1.0 mL/min using a syringe pump (Razel Scientific Instruments model R100-E, Saint

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Albans, Vermont) to ensure surface-controlled reaction rather than diffusion control.36 This

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flow rate does not influence the adsorption of the peptides (1, 50 or 100 nM) on the crystal

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faces. AFM images were collected using Si3N4 tips (Bruker DNP-S10, spring constants of

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0.12-0.35 N/m or ScanAsyst-Fluid + with a spring constant of 0.7 N/m) with scan rates of 2-4

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Hz. Measurements were made on more than three crystals per solution composition to ensure

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reproducibility of the results, and the images were analyzed using the NanoScope analysis

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software.

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In Situ AFM for Self-Assembly and Disassembly of Amel’s C-Terminus Peptides on

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OCP. Pure peptide solutions (1, 50, or 100 nM in 25 mM Tris-HCL buffer, 25 °C, pH 6.5)

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were passed over OCP crystal surfaces and all in situ AFM observations were imaged in

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ScanAsyst mode. Experiments at each peptide concentration were repeated three times.

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Scanning Confocal Interference Microscopy (SCIM). Leica TCS SP8 SCIM (Wetzlar,

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Hesse, Germany) using a helium/neon laser (λ= 632.8 nm) or a krypton/argon laser (λ= 488

149

nm) was used to image the adsorption of FITC labeled Amel’s C-terminal peptides at 1, 50 6


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and 100 nM on the OCP (100) surfaces pre-immersed in a supersaturation solution (σ = 1.98).

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In all cases, a 40 × water-immersion objective and a 90/10 mirror as a beam splitter were used.

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All procedures were carried out in the dark.37

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Single-Molecule Force Spectroscopy (SMFS). Force measurements were made with a

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Bruker MultiMode VIII AFM (Bruker, Santa Barbara, CA) using Si3N4 cantilevers with

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triangular levers (Bruker SNL-10, spring constants of 0.06 N/m) in all force determination

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experiments. Details of AFM tip functionalization can be found in ref.13 In brief, new tips

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were immersed in acetone for 30 min, rinsed in ethanol, and then dried under room

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temperature.13 These cleaned tips were coated with 30 nm Au by thermal evaporation, and

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then were immersed in a N, N-dimethylformamide (DMF) (Sigma, St. Louis, Missouri)

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solution containing 0.2 mM of the heterobifunctional cross-linker LC-SPDP (Thermo

161

Scientific, Waltham, Massachusetts) consisting of a pyridyl disulfide that adsorbs to Au, and

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an N-hydroxysuccinimide (NHS) ester that reacts with the N-terminal residue of the peptide

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through nucleophilic attack to form a stable covalent bond.13 After rinsing in DMF, followed

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by ethanol, the tips were immersed overnight in an Amel’s C-terminal peptide solution at 40

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nM in phosphate buffer solutions (PBS).13 Finally, the peptide was anchored linking to the

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NHS ester-bearing tips in PBS at pH 6.5. It is necessary to use limited concentration of

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peptide for functionalizing tips in order to acquire a single molecule linking due to the

168

possibility of self-assembly of the 13-mer Amel’s C-terminal peptides.13 The functionalized

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tips were rinsed in pure water prior to use.

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Force measurements between modified tips and OCP crystals or mica were performed in

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PBS at pH 6.5 using a Bruker MultiMode VIII AFM (Santa Barbara, CA). Details of force

172

measurements and the theoretical analyses of binding free energies can be found in refs.13,38

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Force curves were measured for each velocity at 256 locations in 2×2 µm2 on the crystal

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surface. 7



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Dynamic Light Scattering (DLS). DLS was performed using a peptide sample that was

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filtered into a flow cell of the instrument Zatasizer Nano ZS90 (Malvern, Worcestershire, UK)

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at room temperature. Peptide solutions at concentrations of 1, 50 or 100 nM were prepared by

178

dissolution of the lyophilized 13-mer Amel’s C-terminal peptides in 10 mM Tris-HCl buffer

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(Aldrich, St. Louis, Mossorui) (IS = 0.15 M, and pH 6.5) in the absence and presence of 4.1

180

mM CaCl2 and stored at 4 °C prior to the experiments.39-41 The data were analyzed by using

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Zetasizer sotfware.

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RESULTS AND DISCUSSION

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Growing OCP Crystals by Attaching Particles in Pure Supersaturated Solutions. We

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used in situ AFM in ScanAsyst mode to observe the OCP (100) growth (Figure 1A) in order

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to minimize the potential dislodgement/removal of particles caused by the movement of the

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AFM tip. At σ = 1.77, AFM images showed that the sizes of spherical particles in both width

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and length gradually increased with reaction times, from 78.12 ± 39.19 nm to 178.31 ±

190

35.34 nm in length; from 79.70 ± 18.62 nm to 167.90 ± 11.24 nm in width after 100 and

191

500 min of growth, respectively (Figure 2B). This demonstrated that the ratio of length over

192

width of forming particles remained at about 1 (Figure 2C). Some aggregated

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pancake-shaped particles formed after 150 min of growth (Figure 1A). When ScanAsyst

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mode was changed to contact mode, these aggregated pancake-shaped particles were

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extensively detected even after 540 min of growth (Figure 1B). The height (about 2.0 nm) of

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the particles grown on the OCP (100) surface within the AFM experimental time frame

197

(0-540 min), regardless of the imaging mode, remained almost constant at different

198

supersaturations ranging from σ = 1.77 to 1.98 (Figure 2A).

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Our real-time observations reveal that the OCP surface growth is through particle 8


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attachment and aggregation in pure supersaturated solutions (Figure 1A and B). Crystals

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grow in a number of ways, including pathways involving the assembly of other particles and

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multi-ion complexes.42 In the present investigations, in situ AFM results revealed that

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primary particles (amorphous or crystalline) exist throughout crystallization processes,

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implying that crystallization by particle attachment (CPA)42-44 is a prevalent growth

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mechanism, especially at early stages of OCP surface crystallization. The consistency of

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particle height further suggests a characteristic of OCP crystallization by the attachment and

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fusion of primary particles with a height of about 2-3 nm, in a good agreement with the

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observation of the heights of the primary particles formed during HAP surface

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crystallization.45

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Elongated Growth in the Presence of Amel’s C-Terminal Peptides. At concentrations of

211

1, 50 and 100 nM Amel’s C-terminal peptides in supersaturated solutions (σ = 1.77, 1.86 and

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1.98), we also observed, at the earliest stages, the formation of stable Ca-P nanoparticles

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with heights of about 2 nm (Figures 3A and 4A). After 360 min, particle elongation with an

214

aspect ratio of about 2:1 occurred in the presence of 50 nM Amel’s C-terminal peptides

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(Figures 3A and 4C). Compared to the presence of 1 or 100 nM Amel’s C-terminal peptides

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(Figures S2 and S3), the particle lengths grown in the presence of 50 nM Amel’s C-terminal

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peptides at σ = 1.77, 1.86 and 1.98 increased to 182.7±46.0 nm, 246.3±93 nm and 298.1±

218

37.4 nm, respectively, whereas the widths remained at 103.8 ±33.1 nm, 114.0 ± 37.0 nm

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and 143.5 ± 25.6 nm, respectively (Figures 4B, S4 and S5). During the growth, the heights

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of particles remained constant at about 2.0 nm at all peptide concentrations and

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supersaturations tested (Figure 4A). Using contact mode, elongated particles became more

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evident after 300 min (Figures 3B, 4C and S6) in all supersaturated solutions containing 50

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nM Amel’s C-terminal peptides.

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The Role of Amel’s C-Terminal Peptides in Particle Attachment. To further understand 9



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how elongated Ca-P particles formed in the presence of Amel’s C-terminal peptides only at

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50 nM, we used in situ AFM to observe the size and morphology of pure Amel’s C-terminal

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peptides adsorbed onto the (100) face of OCP (in the absence of OCP supersaturated

228

solutions). As shown in Figure 5, discrete peptide nanoparticles with heights of about 1.1 nm,

229

3.0-4.0 nm and 2.0-3.0 nm formed at concentrations of 1, 50 and 100 nM, respectively (Figure

230

5D and E). Interestingly, only at 50 nM concentrations, these particles connected to each other

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(as shown within the blue rectangles in Figure 5B2) to form elongated nanorod-like

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assemblies (Figure 5B3 and B4) with an aspect ratio of about 2:1 (Figure 5F). The

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disassembly of relatively large spherical peptide particles was observed at 100 nM (Figure 5C)

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and the height of particles gradually decreased to about 1.2 nm from 3.0 nm while the aspect

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ratio was kept at about 1:1 (Figure 5F). Following the adsorption of 1 nM peptides on the

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OCP (100) surface, no aggregated particles formed (Figure 5A). This was identified by

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fluorescence imaging using SCIM to observe the adsorption of the Amel’s C-terminal

238

peptides modified by FITC on the OCP (100) crystal surfaces (Figure 6), and results showed

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that only at 50 nM, elongated and oriented peptides with green fluorescence were observed

240

(Figure 6B), whereas at 1 nM and 100 nM no elongated peptide assembles on the OCP (100)

241

crystal surface were seen (Figure 6A and C).

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Real-time AFM images as seen in Figure 5B show the adsorption of 50 nM Amel

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peptides on OCP, and then these adsorbed peptides assembled into well-aligned nanorods. The

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height of primary peptide particles (about 2.2 nm) corresponds to the theoretical

245

hydrodynamic radius RH of the dimer and trimer of the 13-mer Amel’s C-termini (26 residues)

246

according to the equation 𝑅3 = 4.75 ± 1.11 𝑁 :.;

In Situ Atomic Force Microscopy Imaging of Octacalcium Phosphate

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