Published: May 13, 2011 r2011 American Chemical Society 7077 dx.doi.org/10.1021/la2006953 | Langmuir 2011, 27, 7077–7083 ARTICLE pubs.acs.org/Langmuir Multistep Growth Mechanism of Calcium Phosphate in the Earliest Stage of Morphology-Controlled Biomineralization Takayuki Nonoyama, † Takatoshi Kinoshita,* ,† Masahiro Higuchi, ‡ Kenji Nagata, ‡ Masayoshi Tanaka, † Kimiyasu Sato, § and Katsuya Kato* ,§ † Department of Frontier Materials and ‡ Department of Materials Science and Engineering, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi 466-8555, Japan § National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimo-Shidami Moriyama-ku, Nagoya, Aichi 463-8560, Japan b S Supporting Information ’ INTRODUCTION Biomineralization is a biological synthesis process for growing hard tissues such as bones, teeth, and shells at ordinary tempera- tures and pressures. The study of biomineralization not only facilitates the clean and efficient industrial synthesis of specific functional materials 1 5 but also helps to clarify fundamental theories of biology. To investigate the essence of biomineralization during nuclea- tion and early crystal growth, we require a mineralization tem- plate that enables us to manipulate or fine tune the arrangement of functional groups on a membrane substrate on an angstrom scale. Previous in vitro studies used templates of a general polymer film or a lipid membrane, and it is difficult to fabricate a template with a functional-groups-arranged surface. Therefore, we chose a simple sequence of peptides for our template. Pep- tides have two main advantages over polymers and lipids. First, the functional-group position on a peptide can be completely determined by self-assembly 6 8 because peptides are known to form stable secondary structures based on their amino acid sequences (their so-called primary structures) and external conditions (such as pH, temperature, and concentration). 9 14 For example, β-sheet conformational peptides are secondary stretch structures of peptide main chains with functional groups derived from amino acid side chains positioned at regular 7 Å intervals. 15 In addition, peptides are known to self-assemble and form a nanofiber by intermolecular hydrogen bonding. 16 18 Second, because a peptide consists of a chain of amino acids, various functional groups derived from its side chains can be readily introduced into the peptide sequence. We previously fabricated a self-assembled monolayer of highly ordered peptide nanofibers using a simple β-sheet peptide poly(ethylene glycol) (peptide PEG) diblock copolymer on a mica substrate. 19 Functional groups of amino acid side chains are arranged at strict 7 Å intervals on the surface of the peptide membrane, and the nucleation site of the peptide part is sepa- rated by PEG on the angstrom scale. We also investigated the effect of the functional-group position on the crystal phase and morphology of a CaP mineralization system grown on the peptide template. 20 We found that highly ordered amorphous CaP (ACP) nanofibers formed. Normally, ACP precipitates do not grow anisotropically and their morphology is difficult to control. 21,22 However, precipitates grown on a peptide template clearly differ in morphology from those grown on a bare mica substrate. On a peptide template, embryos of CaP precipitate, which are less stable because of their small size, are confined in a linear alignment. Although this result is interesting in the con- finement effect induced by the organic substance, the embryos do not have a uniform growth rate. Rather, their growth rate differs Received: February 23, 2011 Revised: April 25, 2011 ABSTRACT: We studied the effect of surface-functional-group position on precipitate morphology in the earliest stage of calcium phosphate biominer- alization and determined the detailed mechanism of precipitation starting from nucleation to precipitate growth. The biomineralization template was a β-sheet peptide scaffold prepared by adsorption with carboxyl groups arranged at strict 7 Å intervals. Phosphate was then introduced. Within 10 s, highly ordered embryos of calcium phosphate were formed and confined by a peptide nanofiber pattern. They repeatedly nucleated and dissolved, with the larger embryos absorbing the smaller ones in a clear demonstration of an Ostwald-ripening-like phenomenon, then aggregated in a line pattern, and finally formed highly ordered nanofibers of amorphous calcium phosphate. This multistep growth process constitutes the earliest stage of biomineralization.