Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite

Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated mechanisms. They have superior mechanical properties due to their complex architecture. Control over biomineral properties can be accomplished by regulation of particle size, shape, crystal orientation, and polymorphic structure. In many organisms, biogenic minerals are assembled using a transient amorphous mineral phase. Here we report that organic constituents of bones and teeth, namely type I collagen and dentin matrix protein 1 (DMP1), are effective crystal modulators. They control nucleation of calcium phosphate polymorphs and the assembly of hierarchically ordered crystalline composite material. Both full-length recombinant DMP1 and post-translationally modified native DMP1 were able to nucleate hydroxyapatite (HAP) in the presence of type I collagen. However, the N-terminal domain of DMP1 (amino acid residues 1-334) inhibited HAP formation and stabilized the amorphous phase that was formed. During the nucleation and growth process, the initially formed metastable amorphous calcium phosphate phase transformed into thermodynamically stable crystalline hydroxyapatite in a precisely controlled manner. The organic matrix-mediated controlled transformation of amorphous calcium phosphate into crystalline HAP was confirmed by x-ray diffraction, selected area electron diffraction pattern, Raman spectroscopy, and elemental analysis. The mechanical properties of the protein-mediated HAP crystals were also determined as they reflect the material structure. Such understanding of biomolecule controls on biomineralization promises new insights into the controlled synthesis of crystalline structures.     

Bone tissue incorporates in vitro gallium with a local structure similar to gallium-doped brushite

During mineral growth in rat bone-marrow stromal cell cultures, gallium follows calcium pathways. The dominant phase of the cell culture mineral constitutes the poorly crystalline hydroxyapatite (HAP). This model system mimics bone mineralization in vivo. The structural characterization of the Ga environment was performed by X-ray absorption spectroscopy at the Ga K-edge. These data were compared with Ga-doped synthetic compounds (poorly crystalline hydroxyapatite, amorphous calcium phosphate and brushite) and with strontium-treated bone tissue, obtained from the same culture model. It was found that Sr²⁺ substitutes for Ca²⁺ in the HAP crystal lattice. In contrast, the replacement by Ga³⁺ yielded a much more disordered local environment of the probe atom in all investigated cell culture samples. The coordination of Ga ions in the cell culture minerals was similar to that of Ga³⁺, substituted for Ca²⁺, in the Ga-doped synthetic brushite (Ga-DCPD). The Ga atoms in the Ga-DCPD were coordinated by four oxygen atoms (1.90 Å) of the four phosphate groups and two oxygen atoms at 2.02 Å. Interestingly, the local environment of Ga in the cell culture minerals was not dependent on the onset of Ga treatment, the Ga concentration in the medium or the age of the mineral. Thus, it was concluded that Ga ions were incorporated into the precursor phase to the HAP mineral. Substitution for Ca²⁺ with Ga³⁺ distorted locally this brushite-like environment, which prevented the transformation of the initially deposited phase into the poorly crystalline HAP.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations ACP amorphous calcium phosphate - DCPD dicalcium phosphate dihydrate (brushite) - HAP hydroxyapatite - ED-XRF energy dispersive X-ray fluorescence - EXAFS extended X-ray absorption fine structure - Ga-ACP gallium-doped amorphous calcium phosphate - Ga-DCPD gallium-doped brushite - Ga-HAP gallium-doped hydroxyapatite - XANES X-ray absorption near edge structure - XAS X-ray absorption spectroscopy - XRD X-ray diffraction     

Chick embryo anchored alkaline phosphatase and mineralization process in vitro.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.     

Differential effects of zinc and magnesium ions on mineralization activity of phosphatidylserine calcium phosphate complexes

Mg²⁺ and Zn²⁺ are present in the mineral of matrix vesicles (MVs) and biological apatites, and are known to influence the onset and progression of mineral formation by amorphous calcium phosphate (ACP) and hydroxyapatite (HAP). However, neither has been studied systematically for its effect on mineral formation by phosphatidylserine-Ca²⁺-Pi complexes (PS-CPLX), an important constituent of the MV nucleation core. Presented here are studies on the effects of increasing levels of Mg²⁺ and Zn²⁺ on the process of mineral formation, either when present in synthetic cartilage lymph (SCL), or when incorporated during the formation of PS-CPLX. Pure HAP and PS-CPLX proved to be powerful nucleators, but ACP took much longer to induce mineral formation. In SCL, Mg²⁺ and Zn²⁺ had significantly different inhibitory effects on the onset and amount of mineral formation; HAP and PS-CPLX were less affected than ACP. Mg²⁺ and Zn²⁺ caused similar reductions in the rate and length of rapid mineral formation, but Zn²⁺ was a more potent inhibitor on a molar basis. When incorporated into PS-CPLX, Mg²⁺ and Zn²⁺ caused significantly different effects than when present in SCL. Even low, subphysiological levels of Mg²⁺ altered the inherent structure of PS-CPLX and markedly reduced its ability to induce and propagate mineral formation. Incorporated Zn²⁺ caused significantly less effect, low (<20 μM) levels causing almost no inhibition. Levels of Zn²⁺ present in MVs do not appear to inhibit their nucleational activity.     

Orientation of bone mineral and its role in the anisotropic mechanical properties of bone--transverse anisotropy

An orientation of hydroxyapatite (HAP) crystals in bovine femur mineral was investigated by means of X-ray pole figure analysis (XPFA). It was found that the c-axis of HAP generally orients parallel to the longitudinal axis of bone (bone axis) and a significant amount of c-axis was oriented in other directions, in particular, perpendicular to the bone axis. Comparing these results with those of the small angle X-ray scattering (SAXS) investigation by Matsushima et al. (Jap. J. appl. Phys. 21, 186-189, 1982) at least two types of morphology of bone mineral were found; rod like bone mineral having the c-axis of HAP crystal parallel to the longitudinal axis of the rod and that having the c-axis not parallel, in a particular case, perpendicular to its longitudinal axis. Transverse anisotropy in mechanical properties of bone was reproduced by the estimation of Young's moduli by using the structural results mainly from XPFA. It is concluded that the anisotropy in mechanical properties of bone is well explained by taking account of the non-longitudinal (off-bone) axial distribution of orientation of bone mineral.     

一株黑曲霉(Aspergillus niger)对羟基磷灰石合成的诱导

【目的】研究利用真菌诱导的方式合成羟基磷灰石(HAP)。【方法】采用含不同浓度Na2HPO4和CaCO3的PDA(Potato Dextrose Agar Medium)液体培养基,研究黑曲霉作用诱导HAP合成,并用透射电镜(TEM)观察诱导形成的矿物晶体形态和结构、用X射线衍射(XRD)法确定矿物种类。【结果】主要研究结果如下:(1)在含有合适浓度的Na2HPO4和CaCO3的PDA液体培养基中,接入黑曲霉可以诱导HAP晶体的合成。(2)黑曲霉对HAP合成的诱导作用跟反应时间有关,反应时间越长,越有利于生成HAP。分析认为黑曲霉对HAP诱导作用是因其代谢产酸造成对CaCO3的溶解以及菌丝体对Ca2+的富集作用,在菌丝球内先形成白磷钙石,然后进一步转化为羟基磷灰石。【结论】黑曲霉在含Na2HPO4和CaCO3的PDA液体培养基中能诱导羟基磷灰石的生成。由于黑曲霉诱导合成HAP的反应条件温和,制备工艺简单,成本低,因此具有潜在应用前景。     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.     

In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca(2+) to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg(2+) and HCO(-)(3) from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 > or = avian cartilage Anx-A6 > cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.     

Role of system Gly in glycine transport in monolayer cultures of liver cells

EXPFS spectra have been recorded from bone mineral and related calcium phosphates. Fourier transformation of a spectrum, using theoretically calculated phase shifts, yields a good approximation to the radial distribution of atoms around a calcium ion. Comparison of the results shows that bone mineral is appreciably different from crystalline synthetic hydroxyapatite and geological apatites, which are similar to each other, but closely resembles hydroxyapatite obtained by maturation of amorphous calcium phosphate.     

Formation of nano-hydroxyapatite in gelatin droplets and the resulting porous composite microspheres

A one-pot strategy was first presented in this paper to synthesize gelatin/hydroxyapatite (HAP) composite microspheres in a water-in-oil (W/O) emulsion. Using gelatin droplets as microreactors and colloid protective medium, needle-like nano-HAP crystals (5 nm x 60-100 nm) in form of clusters were homogeneously and orderly precipitated within gelatin matrix. The results of scanning electron microscopy (SEM) revealed that the as-prepared microspheres with an average diameter of 7.5 microm displayed a narrow particle size distribution, a high dispersity and a naturally porous structure. This microsphere material is expected to have a great potential for both controlled drug release and faster bone in-growth in bone tissue engineering.     

Biomimetic CoUagen/Hydroxyapatite Composite Scaffolds： Fabrication and Characterizations

Biomimetic collagen/hydroxyapatite scaffolds have been prepared by microwave assisted co-titration of phosphorous acid-containing collagen solution and calcium hydroxide-containing solution. The resultant scaffolds have been characterised with respect to their mechanical properties, composition and microstructures. It was observed that the in situ precipitation process could combine collagen fibril formation and hydroxyapatite （HAp） formation in one process step. Collagen fibrils guided hydroxyapatite precipitation to form bone-mimic collagen/hydroxyapatite composite containing both intrafibrillar and interfibrillar hydroxyapatites. The mineral phase was determined as low crystalline calcium-deficient hydroxyapatite with calcium to phosphorus ratio （Ca/P） of 1.4. The obtained 1% （collagen/HAp = 75/25） scaffold has a porosity of 72% and a mean pore size of 69.4 ~tm. The incorporation of hydroxyapatite into collagen matrix improved the mechanical modulus of the scaffold significantly. This could be attributed to hydroxyapatite crystallites in collagen fibrils which restricted the deformation of the collagen fibril network, and the load transfer of the collagen to the higher modulus mineral component of the composite.     

Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds

The most widely accepted hypothesis to account for maturational changes in the X-ray diffraction characteristics of bone mineral has been the 'amorphous calcium phosphate theory', which postulates that an initial amorphous calcium phosphate solid phase is deposited that gradually converts to poorly crystalline hydroxyapatite. Our studies of bone mineral of different ages by X-ray radial distribution function analysis and 31P n.m.r. have conclusively demonstrated that a solid phase of amorphous calcium phosphate does not exist in bone in any significant amount. 31P n.m.r. studies have detected the presence of acid phosphate groups in a brushite-like configuration. Phosphoproteins containing O-phosphoserine and O-phosphothreonine have been isolated from bone matrix and characterized. Tissue and cell culture have established that they are synthesized in bone, most likely by the osteoblasts. Physiochemical and pathophysiological studies support the thesis that the mineral and organic phases of bone and other vertebrate mineralized tissues are linked by the phosphomonester bonds of O-phosphoserine and O-phosphothreonine, which are constituents of both the structural organic matrix and the inorganic calcium phosphate crystals.     

Studies on induction of L-aspartic acid modified chitosan to crystal growth of the calcium phosphate in supersaturated calcification solution by quartz crystal microbalance

Acidic amino acids, such as aspartic acid (L-Asp) and glutamic acid, are the primary bioactive molecules of the glycoprotein on the organic/inorganic interface of biomineralized tissues. In this study, the induction of chitosan film modified with L-Asp on the crystal growth of hydroxyapatite (HAP) was investigated by a novel in situ analysis approach, quartz crystal microbalance (QCM), associated with the dynamically structural and morphological characterization of precipitation products on various phases by X-ray diffraction (XRD), Fourier-transformed infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The natural chitosan exhibited no inducing ability on the crystal growth of HAP. However, the growth rate of induced HAP was dramatically accelerated by the L-Asp modification of chitosan film and increased with the increase of the concentration of L-Asp in the chitosan substrate. It was shown that the chelation of calcium ion with L-Asp provided a nucleation centre and the cluster nuclei was formed by adsorbing further PO(4)(3-), Ca(2+), and then HAP deposited on the original HAP coating in the supersaturated calcification solution (SCS). The developed method allows a kinetic evaluation of the induction of organic film on crystal nucleation and the growth of HAP in vitro.     

Peptides of Matrix Gla Protein Inhibit Nucleation and Growth of Hydroxyapatite and Calcium Oxalate Monohydrate Crystals

Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.     

Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D

In order to generate a calcium-phosphate bone cement as a transient replacement for bone defects, we modified Biocement D (Merck Biomaterial GmbH) containing mineralised collagen with osteocalcin, the most abundant non-collageneous protein of bone. Osteocalcin was added to the cement paste during setting in order to control the crystallisation kinetics of hydroxyapatite (HAP) as well as to stimulate the interaction of osteoblasts and osteoclasts with the bone replacement material. Analysis by SEM and AFM shows, that the addition of osteocalcin causes a nanosize microstructure of the calcium cement, which can be explained by inhibited growth of HAP crystals. The fracture strength of the material decreased by incorporation of osteocalcin, pointing onto a higher defect concentration of the crystalline structure. The impact of osteocalcin onto the interaction of bone cells with HAP-Collagen I-cements was studied in a cell culture system using the human osteosarcoma cell line SAOS-2. Results suggest, that osteocalcin might possibly improve the initial adherence of osteoblast-like cells, whereas proliferation of the cells is not effected.     

Developmental changes in bone mineral structure demonstrated by extended X-ray absorption fine structure (EXAFS) spectroscopy

EXAFS spectra have been recorded above the calcium K edge from bones of mice aged 3 days, 1 week, 1 month, 2 months and 7 months. Spectra indicated that the calcium ion environment in bone mineral changes during development. Results were compared with those obtained from amorphous calcium phosphate and a poorly crystalline hydroxyapatite matured from this amorphous calcium phosphate in the presence of water. Spectra from the older mice closely resembled those of the matured product but those from the younger mice were more like those from the freshly prepared amorphous calcium phosphate.     

Principles of mimicking and engineering the self-organized structure of hard tissues

The mechanism of the formation of a self-aligned hydroxyapatite (HAP) nanocrystallite structure was examined. It is found that the highly ordered HAP nanocrystallite assembly is attributed to the so-called self-(homo)epitaxial nucleation and growth. On the other hand, according to this mechanism, a high supersaturation will give rise to a random assembly of HAP crystallites. The effects of ions, biosubstrate, and supersaturation on the micro/nanostructure correlation between substrate and biominerals as well as their implications in hard tissue formation were examined. Surprisingly, some biomolecules are found to be able to suppress the supersaturation-driven interfacial structure mismatch and hence promote the well aligned HAP pattern formation.     

Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens

Matrix vesicles (MVs) in the growth plate bind to cartilage collagens and initiate mineralization of the extracellular matrix. Native MVs have been shown to contain a nucleational core responsible for mineral formation that is comprised of Mg(2+)-containing amorphous calcium phosphate and lipid-calcium-phosphate complexes (CPLXs) and the lipid-dependent Ca(2+)-binding proteins, especially annexin-5 (Anx-5), which greatly enhances mineral formation. Incorporation of non-Ca(2+)-binding MV lipids impedes mineral formation by phosphatidylserine (PS)-CPLX. In this study, nucleators based on amorphous calcium phosphate (with or without Anx-5) were prepared with PS alone, PS + phosphatidylethanolamine (PE), or PS + PE and other MV lipids. These were incubated in synthetic cartilage lymph containing no collagen or containing type II or type X collagen. Dilution of PS with PE and other MV lipids progressively retarded nucleation. Incorporation of Anx-5 restored nucleational activity to the PS:PE CPLX; thus PS and Anx-5 proved to be critical for nucleation of mineral. Without Anx-5, induction of mineral formation was slow unless high levels of Ca(2+) were used. The presence of type II collagen in synthetic cartilage lymph improved both the rate and amount of mineral formation but did not enhance nucleation. This stimulatory effect required the presence of the nonhelical telopeptides. Although type X collagen slowed induction, it also increased the rate and amount of mineral formation. Both type II and X collagens markedly increased mineral formation by the MV-like CPLX, requiring Anx-5 to do so. Thus, Anx-5 enhances nucleation by the CPLXs and couples this to propagation of mineral formation by the cartilage collagens.     

Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.