Published: June 09, 2011 r2011 American Chemical Society 8263 dx.doi.org/10.1021/la201361e | Langmuir 2011, 27, 8263–8268 ARTICLE pubs.acs.org/Langmuir Cooperative Calcium Phosphate Nucleation within Collagen Fibrils Diana N. Zeiger, † William C. Miles, † Naomi Eidelman, ‡,§ and Sheng Lin-Gibson* ,† † Polymers Division and ‡ Paffenbarger Research Center, American Dental Association Foundation, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8543, United States ’ INTRODUCTION Mineralized tissues such as bones and teeth have hierarchical structures, and when these tissues are damaged, they may require grafts to aid healing. Although effective, grafts have significant drawbacks such as availability, pain at the harvest site, and infectious disease transfer. Thus, synthetic grafts are desirable for the treatment of damaged or diseased mineralized tissues. However, because of the complex structures of these tissues, the formation of synthetic grafts is a problem with significant materials science challenges. 1 Current approaches have sought to mimic biological processes to form these structures. However, despite decades of research, the precise mechanism by which bones and teeth mineralize has yet to be elucidated. A complete understanding of this mechanism has profound implications for treatments for diseases of mineralized tissue, including osteo- porosis, caries, and osteogenesis imperfecta. The key components of bone and dentin are similar and consist of type I collagen and hydroxyapatite 2 assembled such that crystals of apatite are positioned within the collagen fibrils (intrafibrillar mineral) with their c axis aligned along the fibrils. 3À5 The mineral is aligned in such a fashion that the degree of mineralization is not the only factor responsible for the strength and toughness of mineralized tissues. Indeed, intrafibrillar mineralization has been found to be more important for the mechanical properties of bone 6 and dentin than overall mineral content. 7 Chaperone molecules (generally proteins bearing regions rich in acidic moieties) appear to be necessary to the biomineraliza- tion process. 8,9 These proteins belong to a group known as the SIBLING (small integrin-binding, N-linked glycoprotein) family. 10 Carboxylate-rich regions on such SIBLING proteins as dentin and bone sialoprotein have been shown to facilitate the mineralization of collagen 11 and remineralize dentin. 12 Acidic macromolecules such as poly(acrylic acid) (pAA) have been shown to induce the formation of intrafibrillar calcium carbonate, 13 and poly(aspartic acid) (pAsp) has been shown to stimulate the formation of intrafibrillar calcium carbonate 14 and calcium phosphate. 15,16 Several mechanisms or partial mecha- nisms have been proposed within the last several years. 11,14,17,18 Recent work by Sommerdijk et al. utilized cryo-TEM to provide significant insight into the mechanism of biomineralization. 19,20 These studies have shown the importance of prenucleation clusters in the biomineralization process. 20 Additionally, results from cryo-TEM studies convincingly demonstrated the specific region on the collagen fibril at which the infiltration of pAsp- mediated prenucleation clusters occurs. 19 The infiltration pro- cess by stabilized ion clusters is proposed to be charge-driven. This research was designed to isolate the key components of the mineralization process and to understand how the interaction between collagen and the chaperone molecule affects mineraliza- tion. This was achieved by varying the order of addition of the mineralization components (i.e., collagen, Ca/P, pAsp). We utilized SEM and FTIR reflectance microspectroscopy (FTIR- RM) to determine the type, quantity, distribution, and phase of the minerals formed on and in collagen. We also used a model heterobifunctional compound to probe the interaction between ÀCOOH and collagen. We show that through pretreatment of the collagen fibers with pAsp, intrafibrillar mineralization can be slowed or even eliminated. This indicates that the interaction of Received: February 11, 2011 Revised: May 31, 2011 ABSTRACT: Although “chaperone molecules” rich in negatively charged residues (i.e., glutamic and aspartic acid) are known to play important roles in the biomineralization process, the precise mechanism by which type I collagen acquires intrafibrillar mineral via these chaperone molecules remains unknown. This study demonstrates a mechanism of cooperative nucleation in which three key components (collagen, chaperone molecules, and Ca 2þ and PO 4 3À ) interact simultaneously. The mineralization of collagen under conditions in which collagen was exposed to pAsp, Ca 2þ , and PO 4 3À simultaneously or pretreated with the chaperone molecule (in this case, poly(aspartic acid)) before any exposure to the mineralizing solution was compared to deduce the mineralization mechanism. Depending on the exact conditions, intrafibrillar mineral formation could be reduced or even eliminated through pretreatment with the chaperone molecule. Through the use of a fluorescently tagged polymer, it was determined that the adsorption of the chaperone molecule to the collagen surface retarded further adsorption of subsequent molecules, explaining the reduced mineralization rate in pretreated samples. This finding is significant because it indicates that chaperone molecules must interact simultaneously with the ions in solution and collagen for biomimetic mineralization to occur and that the rate of mineralization is highly dependent upon the interaction of collagen with its environment.