In Situ Atomic Force Microscopy Imaging of Octacalcium Phosphate

Mar 21, 2017 - (18) A combination of the single-molecule force and molecular simulations has demonstrated that the C-terminal fragment exhibits a high...
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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

13 14

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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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,

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

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

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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

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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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Crystal Growth & Design

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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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Crystal Growth & Design

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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

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

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Crystal Growth & Design

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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

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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

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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

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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

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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

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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 ±

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

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

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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

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

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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

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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

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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±

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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

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

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

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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

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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

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

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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

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

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