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Amelogenin affects brushite crystal morphology and promotes its phase transformation to monetite Dongni Ren, Qichao Ruan, Jinhui Tao, Jonathan Lo, Steven Nutt, and Janet Moradian-Oldak Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00569 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 8, 2016
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Amelogenin affects brushite crystal morphology and
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promotes its phase transformation to monetite
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Dongni Ren1, Qichao Ruan1, Jinhui Tao2, Jonathan Lo3, Steven Nutt3and Janet Moradian-
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Oldak1*
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1 Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, University of Southern
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California, Los Angeles, CA 90033, USA, 2 Physical Sciences Division, Pacific Northwest National Laboratory,
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Richland, WA 99352, USA, 3 Mork Family Department of Chemical Engineering and Materials Science, University
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of Southern California, Los Angeles, CA, USA
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*Corresponding author: Janet Moradian-Oldak, Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033, USA. email:
[email protected], Tel: 323-442-1759, Fax: 323-442-2981
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ABSTRACT
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Amelogenin protein is involved in organized apatite crystallization during enamel formation.
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Brushite (CaHPO4·2H2O), which is one of the precursors of hydroxyapatite mineralization in
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vitro, has been used for fabrication of biomaterials for hard tissue repair. In order to explore its
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potential application in biomimetic material synthesis, we studied the influence of the enamel
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protein amelogenin on brushite morphology and phase transformation to monetite. Our results
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show that amelogenin can adsorb onto the surface of brushite, leading to the formation of layered
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morphology on the (010) face. Amelogenin promoted the phase transformation of brushite into
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monetite (CaHPO4) in the dry state, presumably by interacting with crystalline water layers in
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brushite unit cells. Changes to the crystal morphology mediated by amelogenin continued even
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after the phase transformation from brushite to monetite, leading to the formation of organized
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platelets with an interlocked structure. This effect of amelogenin on brushite morphology and the
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phase transformation to monetite could provide a new approach to developing biomimetic
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materials.
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INTRODUCTION
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Enamel is the outermost layer of the mammalian tooth and is considered to be one of the hardest
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calcium phosphate-containing bioceramics.1 This calcified tissue is comprised of more than 85
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v/v% (or 98 wt %) hydroxyapatite (HA) crystals and, being closely connected to the underlying
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dentin, shows exceptional biological and mechanical properties.2,3 The high wear- and fracture-
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resistance of enamel is associated with the highly ordered structure of enamel prisms (rods) and
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the interwoven arrangement of HA that produces inter-prismatic enamel.4-6 The formation of the
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basic building blocks of enamel, long and thin HA crystals, is mediated by the components of the
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extracellular matrix secreted by the ameloblast cells.7 Among the matrix proteins, amelogenin is
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the most prevalent and is essential for normal enamel formation.8,9 It is well accepted that
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amelogenin plays critical roles in regulating enamel crystal morphology and organization
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through its specific interaction with forming enamel crystals. In order to get better insight into
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the function of amelogenin in mediating mineral formation in enamel, investigators have used in
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vitro models to study interactions between amelogenin and calcium phosphate crystals, including
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hydroxyapatite and octacalcium phosphate.10-12 In contrast, there has been little study of how
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amelogenin may mediate brushite (dicalcium phosphate dehydrate, CaHPO4·2H2O) or monetite
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(dicalcium phosphate anhydrate, CaHPO4) crystal growth.
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Brushite and monetite are metastable phases of calcium orthophosphate. Brushite crystals consist
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of parallel Ca-PO4 chains that are held together by lattice water molecules via hydrogen bonds.13
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The loss of crystal water results in the transformation from brushite into anhydrous monetite.
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The complete dehydration of brushite usually requires heating at a relatively high temperature,
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over 200 °C.14 Owing to their thermodynamic metastability under physiological conditions, 15-17
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both brushite and monetite have been considered suitable for developing calcium phosphate
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biomaterials.18-20 Several studies on the application of brushite for fabricating cements for bone
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and tooth repair have been reported. Kumar et al. used single brushite crystals as seeds for the
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growth of hydroxyapatite layers as implant materials.21 Biemond et al. reported that a biomimetic
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brushite coating on an implant enhanced bone ingrowth significantly. 22 There have been several
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reports on biomimetic approaches for bone tissue engineering using monetite together with
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hydroxyapatite. Hsu et al. reported the growth of monetite crystals with diverse morphologies on
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top of hydroxyapatite under hydrothermal conditions.23 Using a liquid crystal matrix and the
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concept of a mineral bridge, He et al. successfully synthesized monetite with superstructures
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similar to the mineral bridge in natural biominerals.24 While biomaterials with potential
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application in hard tissue repair have been developed, in most of these reports extreme
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conditions far from those of physiological environments were used, including high temperatures
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(above 200 °C) and/or extreme pH values.
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Brushite and monetite are effective starting materials in the preparation of various nanostructured
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hydroxyapatite crystals.25, 26 For example, nanoneedles, fibers, and sheets of hydroxyapatite have
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been prepared by the hydrolysis of monetite in alkali solutions by varying the pH and ion
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concentrations. Using a single monetite crystal as a template, enamel-like hydroxyapatite (HAp)
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was obtained in an alkaline aqueous solution with the assistance of microwave irradiation27 or
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mediation by organic molecules.28 Recently, we have successfully synthesized a layered
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monetite-chitosan composite that can further transform into a HAp composite with a multilevel
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ordered structure.29 These studies demonstrated that the structural control of brushite and
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monetite precursors by organic molecules is important in the construction of multilevel 3 ACS Paragon Plus Environment
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hierarchical HAp.
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In the present work, considering the role of the amelogenin protein in controlling in vitro calcium
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phosphate crystal formation and its potential application in biomaterial synthesis, we studied the
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effect of amelogenin on brushite crystal morphology and the subsequent phase transformation to
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monetite. We demonstrate that in a dry state, at mild temperature and pH conditions, and in the
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presence of amelogenin, brushite transformed to monetite. Understanding the underlying
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mechanism of phase transformation and the unique structure of monetite derived from phase
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transformation provides us with insights that could lead to a new biomimetic approach to
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developing biomaterials for dental repair needs.
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EXPERIMENTAL SECTION
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Protein Expression and Purification. Recombinant porcine amelogenins (rP172 and rP148)
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were expressed in Escherichia coli strain BL21-codon plus (DE3-RP, Agilent Technologies, Inc.,
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Santa Clara, CA) as previously described.30-33 Protein purification was accomplished on a reverse
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phase C4 column (10 × 250 mm, 5µm) mounted on a Varian Prostar HPLC system
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(ProStar/Dynamics6, version 6.41 Varian, Palo Alto, CA), using a linear gradient of 60%
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acetonitrile at flow rate of 1.5 ml/min. Recombinant porcine amelogenin rP172 is an analog of
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full-length native porcine P173, which has 173 amino acids, but rP172 lacks the N-terminal Met
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and a phosphate group on Ser16.34 The recombinant rP148 lacks the hydrophilic C-terminal. The
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C-terminal (M149-D173) 25 amino acid residue peptide was synthesized at the Microchemical
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Core Laboratory at the University of Southern California, using a Pioneer peptide synthesizer
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(Applied Biosystems, Foster City, CA) following the N-Fmoc-l-aminoacid pentafluorophyenyl
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ester/HOBt coupling method.35
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Cleavage of rP172 by MMP-20. The recombinant human enamel proteinase Matrix
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Metalloproteinase 20 (rhMMP-20, Enzo Life Sciences, Farmingdale, NY) was mixed with 1.0
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mg/ml rP172 at a mole ratio of 1:500 (0.02 µg/µl MMP-20, 5 mM CaCl2 and 20 µM ZnCl2 in 50
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mM Tris-HCl, pH=8.0), then incubated at 37 °C for 18 h during cleavage. The protein mixture
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after cleavage was frozen at -20 °C for subsequent SDS-PAGE and crystallization experiments.
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Electrophoresis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was
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carried out with a 12% acrylamide gel to monitor the progress of amelogenin digestion. The gel 4 ACS Paragon Plus Environment
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was stained with Coomassie brilliant blue. Ten µl of amelogenin cleavage product was loaded at
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a concentration of 1.0 mg/ml. The same volumes and concentrations of rP172 and rP148 were
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also loaded as controls to identify the composition of amelogenin cleavage products. About 5 µl
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Precision Plus ProteinTM KaleidoscopeTM Standard (250 kDa, BIO-RAD, USA) was loaded as
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a marker.
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Amelogenin rP172 Fluorescent Labeling. To observe the distribution of recombinant
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amelogenin rP172 on the crystal surfaces under fluorescence spectroscopy, rP172 was labeled
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with fluorescein isothiocyanate (FITC) as follows: FITC (1.25 µl of 1.0 mg/ml) was mixed with
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rP172 (62.5 µl 1.0 mg/ml) at a 1:50 ratio of FITC to protein at 37 °C in the dark for 1 h.
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Deionized water was used as a control in fluorescent labeling, with the same volume (62.5 µl)
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mixed with FITC (1.25 µl of 1.0 mg/ml) using the same procedure. The FITC-labeled solutions
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were then mixed with the solutions used in the synthesis of brushite crystals.
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Brushite Synthesis with and without Amelogenin. Brushite crystals were synthesized
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following the previously published protocol with a slight modification.36 KH2PO4-CaCl2 solution
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containing rP172 was prepared by adding 62.5 µl of 1.0 mg/ml rP172 into a solution of 2.5 ml
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KH2PO4 (0.18 M) and 1.25 ml CaCl2 (0.4 M). Then, 2.5 ml Na2HPO4 (0.02 M) was slowly
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titrated into the rP172-containing KH2PO4-CaCl2 solution at room temperature, while the pH of
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the reaction solution was maintained at 5.5 by adding NaOH. The final concentration of rP172 in
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the mineralization solution was 0.1 mg/ml. The crystals were allowed to grow at room
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temperature for 5 days while being shaken. The deposited crystals were then separated from the
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solution using a centrifuge at 10,000 rpm for 10 min.
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Sequential Washing of Brushite Crystals. Crystals were prepared for characterization with
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XRD, Fluorescent Microscopy, AFM, and Raman spectroscopy as follows: After centrifuging,
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the crystals were washed sequentially with deionized water (10 times) and phosphate buffer (pH
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5.5, 1M, 30 min), and the samples were centrifuged again at 10,000 rpm for 15 min after each
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wash process. Finally, the precipitates were lyophilized and stored at 4 °C for future
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characterizations. To observe the effect of the washing process, the products were also collected
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without any washing and after washing with only water. Parallel experiments with BSA (at the
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same final concentration of 0.1 mg/ml) or deionized water (containing FITC) were carried out
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under the same conditions for comparison. These are referred to as the BSA control group and
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water control group, respectively. 5 ACS Paragon Plus Environment
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Fluorescence Spectroscopy. Fluorescence spectroscopy was performed using a PTI Quanta
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Master QM-4SE spectrofluorometer (PTI, Birmingham, NJ, USA). For all the samples, the
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fluorescent signal was excited at 435 nm and the emission spectral peak was at 519 nm. The
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same samples were also characterized by confocal microscopy (Leica TCS SP5 confocal
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microscope).
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X-Ray Diffraction (XRD). The lyophilized crystal powder samples were tightly pressed into
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SiO2 diffraction plates 2 mm in height and 20 mm in diameter. The SiO2 Zero Diffraction Plates
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30x30x2.5 mm (2sp) with Cavity 20 ID x 1.0 mm were purchased from MTI Corporation, USA.
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XRD patterns were recorded on a Rigaku Diffractometer with Cu Kα radiation (λ = 1.542 Å)
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operating at 70 kV and 50 mA with a step size of 0.08°, at a scanning rate of 4 °/min with a 2θ
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range from 5° to 65°.
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Fourier Transform Infrared Spectroscopy (FTIR). Transmittance infrared spectroscopy was
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carried out using lyophilized crystal samples/KBr tablets on a Spectrum GX FTIR (Perkin-
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Elmer, USA) in the region of 4000−400 cm−1 with a resolution of 2 cm-1.
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Thermal Gravity Analysis (TGA). Two TGA experiments were conducted separately in this
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study. For Figure 4a, the fresh samples prepared with 0.1 mg/ml amelogenin (experimental
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group) and without protein (control group) were lyophilized and used. For TGA curves in Figure
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5a, the samples prepared with 0.1 mg/ml amelogenin were tested after being kept in dry
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conditions at room temperature for three months. The samples weighed approximately 5 mg, and
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were loaded into tin containers and tightly folded to remove air; the tin containers’ weight was
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pre-balanced. TGA was performed using TA Instruments Q5000 IR equipment. The temperature
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was kept at 25 °C for 10 min, then ramped to 1000 °C at a rate of 50 °C/min, and the sample
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weight loss during the rise in temperature was recorded.
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Scanning Electron Microscopy (SEM). Scanning electron microscopy (SEM) was performed
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using a JEOL 7001-FEG microscope with an accelerating voltage of 10 kV. Sample powder was
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evenly distributed onto adhesive carbon tapes that were attached to aluminum mounts (12 mm in
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diameter, 10 mm in height) and then coated with 80/20 platinum/palladium before loading into
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the microscope.
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Micro-Raman Spectroscopy. Samples were put onto glass cover slips in the form of powder.
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The Raman spectra were collected on single crystals from 100 to 4000 cm-1 under back scattering
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geometry by a LabRAM ARAMIS confocal Raman Microscope (HORIBA scientific, Japan), 6 ACS Paragon Plus Environment
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operated at a resolution of 2 cm-1 with an excitation wavelength of 532 nm and laser power of
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2.5 mW. A ×60 objective with numerical aperture of 0.75 was used to focus the sample and
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collect spectra for 20 seconds. The experiment was repeated 3 times for each sample and results
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were averaged.
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Atomic Force Microscopy (AFM). A muscovite mica disc (9.9 mm in diameter; Ted Pella, Inc.)
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was freshly cleaved and used as a supporting surface. The mica surface was functionalized
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through treatment with 50 µl poly-L-lysine solution (0.1% w/v, Ted Pella) for 5 minutes, then
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thoroughly rinsed with water and dried using a stream of nitrogen gas. The above-mentioned
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freshly prepared and lyophilized DCPD crystals washed with phosphate buffer were dispersed in
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PBS solution (Sigma-Aldrich) containing 10 mM phosphate and 154 mM NaCl at pH 7.4. 20 µl
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of this suspension was placed on the functionalized mica in 1 min. After allowing 10 minutes for
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thermal relaxation, time-lapse AFM images were continuously collected, first in PBS solution
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and later in water inside a fluid cell.
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All in situ AFM images were captured in tapping mode at room temperature (23°C) with a
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NanoScope 8 Atomic Force Microscope (E scanner, Bruker) using silicon tips on silicon nitride
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cantilevers (HYDRA triangular lever, k=0.088 N/m, tip radius
