Chemical analysis of silica doped hydroxyapatite biomaterials consolidated by a spark plasma sintering method

Silica (SiO(2)) and the silicate-based biomaterials play an important role due to their in vitro and in vivo biological response. The present study synthesized a novel nano-structured amorphous silica doped hydroxyapatite (HA) via an aqueous precipitation route. HA was prepared with 0, 1, 3 and 5 wt% silica, which are comparable to the measured silicon content of natural bone. After spray drying into micron sized powders, the silica doped HA (SiHA) powders were consolidated at 1000 degrees C with a dwell time of 3 min using a spark plasma sintering (SPS) technique. X-ray diffraction analysis showed a main apatite phase with minor secondary beta-tricalcium phosphate (beta-TCP) was observed in the as-consolidated SiHA compacts. Substitution of PO(4)(3-) by SiO(4)(4-) in the apatite structure resulting in a small increase in the lattice parameters in both a-axis and c-axis of the unit cell were identified by X-ray photoelectron spectrometer (XPS) analysis and Raman spectrometer investigation. The cell culture in vitro investigation demonstrated that the presence of silicon in the SPS consolidated compacts contributed to the relatively high cell proliferation ability when compared with phase pure HA.     

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the nu(4) PO(4)(3-) vibration.

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.     

An infrared study of the interaction of polymethyl methacrylate with the protein and mineral components of bone.

We used Fourier transform infrared (FT-IR) microscopic mapping techniques to investigate the infiltration of polymethyl methacrylate (PMMA), a widely used medium for embedding biological tissues, into rat femur sections. Monitoring of the infrared absorbances of the PMMA carbonyl stretch, the protein amide I, and the apatite mineral phosphate stretch over a 225 x 975-microns region of the epiphyseal growth plate region of the rat femur enabled comparison of the relative amount of each component in distinct regions of the tissue. It was found that PMMA penetrates less into regions of greater mineral density and that the frequency of the PMMA carbonyl absorbance from the embedded tissue, 1729 cm-1, is identical to the free PMMA carbonyl frequency. This is consistent with a diffusion mechanism of infiltration of the PMMA, with no specific chemical interaction between the PMMA and the tissue components.     

Overgrowths of large authigenic apatite crystals on the surface of conodonts from Cantabrian limestones (Spain)

Conodonts from the middle to upper Paleozoic limestones of the Cantabrian zone commonly show apatite overgrowths. A large crystal microtexture observed under the SEM corresponds to local rims of euhedral to subhedral apatite crystals, which were preceded by the neoformation of smaller crystals. Four types of this microtexture (blocky, columnar, fan, and denticular) are described on different areas of the oral surface of conodonts, whereas dissolution features may be present in the basal cavity area. The distribution of these types of microtexture in different areas of conodont morphology suggests a general trend to neocrystallization, where crystal size increases towards the top of the conodont ornamentation and a chemical gradient controls the crystalline growth. This arrangement is widely related to the surface morphology and to the general conodont histology. The large crystal microtexture grows during early diagenesis from near surface to moderate burial and is linked to the known secondary apatite cement present in natural fused clusters of conodonts. The features described here are also compared to microtextures developed on conodonts during low- to medium-grade metamorphic conditions, where phosphate in solution is available.     

Two-dimensional vibrational correlation spectroscopy of in vitro hydroxyapatite maturation

Two-dimensional (2-D) Raman and 2-D IR correlation spectroscopy are applied to analyze changes in the nu(4) region of the IR spectrum and in the nu(1) region of the Raman spectrum during the maturation of hydroxyapatite (HA) following the solution-mediated conversion of amorphous calcium phosphate (ACP) to HA. The nu(1) region of the Raman spectrum exhibits a frequency shift and sharpening during the maturation. Comparison of the experimental and simulated 2-D plots for this process suggests that the shift of a single peak, rather than a change in the relative intensity of two overlapped bands, is responsible for the observed spectral changes. The nu(4) mode of the PO(3-)(4) ion (T(2) symmetry in the free species) splits into a triplet with components near 563, 575, and 603 cm(-1) in HA. In addition, broad features appear at 540 and 617 cm(-1). During the latest stages of the maturation, an OH(-) librational mode develops at approximately 632 cm(-1). Changes in the relative intensities of three components of the nu(4) mode are not all correlated with each other. The synchronous 2-D plots reveal that the 563 and 603 cm(-1) pair are positively correlated while the feature at 575 cm(-1) is absent. A 587 cm(-1) mode arising from ACP is negatively correlated with the 563 and 603 cm(-1) pair and is both synchronously (positively) and asynchronously correlated with the 540 cm(-1) feature during the early stages of the maturation but is absent from 2-D plots of the later stages of the maturation. Cross correlations between the nu(4) mode and the nu(1),nu(3) contour generally confirm and extend previous assignments for the latter spectral region. Finally, the suitability of the 2-D approach for analysis of IR spectral images is examined through studies of HA crystallinity in a human iliac crest biopsy sample. Trabecular bone contains a fraction of HA that is more crystalline and mature than could be achieved in vitro during the room temperature ACP --> HA interconversion.     

Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

Osteotropic Peptide that differentiates functional domains of the skeleton

HPMA copolymer-d-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.     

Effect of sodium polyacrylate on the hydrolysis of octacalcium phosphate

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of sodium polyacrylate (NaPA). In the absence of the polyelectrolyte, OCP undergoes a complete transformation into HA in 48 h. The hydrolysis is inhibited by the polymer, which is significantly adsorbed on the crystals, up to about 22 wt.%. A polymer concentration of 10(-2) mM is sufficient to cause a partial inhibition of OCP to HA transformation, which is completely hindered at higher concentrations. The small platelet-like crystals in the TEM images of partially converted OCP can display electron diffraction patterns characteristic either of OCP single crystals or of polycrystalline HA, whereas the much bigger plate-like crystals exhibit diffraction patterns characteristic of OCP single crystals. The polyelectrolyte adsorption on OCP crystals is accompanied by an increase of their mean length and by a significant reduction of the coherence length of the perfect crystalline domains along the c-axis direction. It is suggested that the carboxylate-rich polyelectrolyte is adsorbed on the hydrated layer of the OCP (100) face, thus inhibiting its in situ hydrolysis into HA.     

Dimethyl sulfoxide-induced hydroxyapatite formation: a biological model of matrix vesicle nucleation to screen inhibitors of mineralization

To elucidate the inhibition mechanisms of hydroxyapatite (HA), a biological model mimicking the mineralization process was developed. The addition of 4% (v/v) dimethyl sulfoxide (DMSO) in synthetic cartilage lymph (SCL) medium containing 2 mM calcium and 3.42 mM inorganic phosphate (P_i) at pH 7.6 and 37 °C produced HA as matrix vesicles (MVs) under physiological conditions. Such a model has the advantage of monitoring the HA nucleation process without interfering with other processes at the cellular or enzymatic level. Turbidity measurements allowed us to follow the process of nucleation, whereas infrared spectra and X-ray diffraction permitted us to identify HA. Mineral formation induced by DMSO and by MVs in the SCL medium produced crystalline HA in a similar manner. The nucleation model served to evaluate the inhibition effects of ATP, GTP, UTP, ADP, ADP-ribose, AMP, and pyrophosphate (PP_i). Here 10 μM PP_i, 100 μM nucleotide triphosphates (ATP, GTP, UTP), and 1 mM ADP inhibited HA formation directly, whereas 1 mM ADP-ribose and 1 mM AMP did not. This confirmed that the PP_i group is a potent inhibitor of HA formation. Increasing the PP_i concentration from 100 μM to 1 mM induced calcium pyrophosphate dihydrate. We propose that DMSO-induced HA formation could serve to screen putative inhibitors of mineral formation.     

Control of Vertebrate Skeletal Mineralization by Polyphosphates

Background

Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO₃ ⁻)_n) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization.

Principal Findings/Methodology

The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO₄ ³⁻) concentration while permitting the accumulation of a high total PO₄ ³⁻ concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO₄ ³⁻ and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4′,6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation.

Conclusions/Significance

We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.     

Synthesis and in vitro behavior of β-TCP zirconia/polymeric biocomposites for bio-applications

Novel biocomposites were fabricated by impregnating β-tricalcium phosphate (β-TCP)/zirconia particles into the polymers matrix. The composite materials were characterized using thermo-gravimetric analysis (TGA), X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR) analyzes and Scanning Electron Microscopy (SEM). The results confirmed the conversion of hydroxyapatite (HA) to β-TCP at a sintering temperature of 1150 °C with or without zirconia powder. The in vitro behavior was assessed via measurement of calcium and phosphorus ions in SBF (simulated body fluid). FT-IR and SEM of the composites were performed pre and post immersion in SBF. The results prove that the bone like apatite layer formation was enhanced on the β-TCP-Z20/polymeric composite surface more than that on the β-TCP-Z10/polymeric composite. Therefore, the data confirmed that zirconia plays an important role in the enhancement of the apatite formation. The conclusions proved that the β-TCP-Z20/polymeric biocomposites, containing 20% of zirconia, are promising for bone remodeling applications.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

A crystallographic investigation of citrate synthase from pig and chicken heart muscle

Citrate synthase (EC 4. 1. 3. 7.) from pig heart and chicken heart muscle can be crystallized from 10 mM phosphate buffer (pH 7. 0–7. 5) and 10–15% PEG4000 solution. The space group is P4₁2₁2 with one subunit/asymmetric unit for the pig heart enzyme. The chicken heart citrate synthase purified from Blue-dextran as well as ATP-Sepharose affinity chromatography both crystallize in space group P2₁2₁2 with one molecule/asymmetric unit, but they have different unit cell dimensions. The a-axis differs by about 17 Å between these two crystal forms, while b- and c-axis dimensions are virtually identical.     

Comparative analysis of crystallinity changes in cellulose I polymers using ATR-FTIR, X-ray diffraction, and carbohydrate-binding module probes

Cotton fiber cellulose is highly crystalline and oriented; when native cellulose (cellulose I) is treated with certain alkali concentrations, intermolecular hydrogen bonds are broken and Na-cellulose I is formed. At higher alkali concentrations Na-cellulose II forms, wherein intermolecular and intramolecular hydrogen bonds are broken, ultimately resulting in cellulose II polymers. Crystallinity changes in cotton fibers were observed and assigned using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and X-ray diffraction (XRD) subsequent to sodium hydroxide treatment and compared with an in situ protein-binding methodology using cellulose-directed carbohydrate-binding modules (CBMs). Crystallinity changes observed using CBM probes for crystalline cellulose (CBM2a, CBM3a) and amorphous cellulose (CBM4-1, CBM17) displayed close agreement with changes in crystallinity observed with ATR-FTIR techniques, but it is notable that crystallinity changes observed with CBMs are observed at lower NaOH concentrations (2.0 mol dm(-3)), indicating these probes may be more sensitive in detecting crystallinity changes than those calculated using FTIR indices. It was observed that the concentration of NaOH at which crystallinity changes occur as analyzed using the CBM labeling techniques are also lower than those observed using X-ray diffraction techniques. Analysis of crystallinity changes in cellulose using CBMs offers a new and advantageous method of qualitative and quantitative assessment of changes to the structure of cellulose that occur with sodium hydroxide treatment.     

多孔beta-磷酸三钙与磷灰石复合材料的制备及其细胞相容性研究

综合运用三维凝胶叠层法和发泡法制备了多孔β-磷酸三钙支架。将多孔支架在1．5倍模拟体液中浸泡14天,得到材料1;或者将其在氢氧化钠溶液中浸泡4天,再在1．5倍模拟体液中浸泡14天,得到材料2。测定了两种材料的物理性能,讨论了类骨磷灰石层对材料矿物组成及其显微结构等的影响。将两组材料分别与成骨前体细胞在体外复合培养,观察和测定了细胞的形态和增殖情况。结果表明复合材料的主要成分为β-磷酸三钙,表面具有结构不完整的含有碳酸磷灰石的类骨磷灰石,成骨细胞能在两组材料上正常粘附和增殖,而且材料2上的细胞粘附情况更好,说明多孔β-磷酸三钙与磷灰石的复合材料有望成为一种有应用前景的骨修复材料和骨组织工程支架材料。     

Cementum protein 1‐derived peptide (CEMP 1‐p1) modulates hydroxyapatite crystal formation in vitro

A cementum protein 1‐derived peptide (CEMP1‐p1) consisting of 20 amino acids from the CEMP1's N‐terminus region: MGTSSTDSQQAGHRRCSTSN, and its role on the mineralization process in a cell‐free system, was characterized. CEMP1‐p1's physicochemical properties, crystal formation, and hydroxyapatite (HA) nucleation assays were performed. Crystals induced by CEMP1‐p1 were analyzed by scanning electron microscopy, Fourier‐transform infrared spectroscopy‐attenuated total reflectance (FTIR‐ATR), X‐ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM), and atomic force microscopy. The results indicate that CEMP1‐p1 lacks secondary structure, forms nanospheres that organize into three‐dimensional structures, possesses affinity to HA, and induces its nucleation. CEMP1‐p1 promotes the formation of spherical structures composed by densely packed prism‐like crystals, which revealed a Ca/P ratio of 1.56, corresponding to HA. FTIR‐ATR showed predominant spectrum peaks that correspond and are characteristic of HA and octacalcium phosphate (OCP). Analysis by XRD indicates that the crystals show planes with a preferential crystalline orientation for HA and for OCP. HRTEM showed interplanar distances that correspond to crystalline planes of HA and OCP. Crystals are composed by superimposed lamellae, which exhibit epitaxial growth, and each layer of the crystals is structured by nanocrystals. This study reveals that CEMP1‐p1 regulates HA crystal formation, somehow mimicking the in vivo process of mineralized tissues bioformation.     

Calcium environment in encrusting deposits from urinary catheters investigated by interpretation of EXAFS spectra

Extended x-ray absorption fine structure (EXAFS) spectra were recorded, above the K absorption edge of Ca, from 10 encrusted urinary (Foley) catheters obtained from 10 different patients. The presence of poorly crystalline apatite was demonstrated in all the deposits, even though this phase could not be detected in the x-ray powder diffraction patterns of most samples because of the obscuring effect of struvite, which was also present. Furthermore, the EXAFS spectra could be interpreted to yield quantitative information on the average short-range structure surrounding the Ca2+ ions in the poorly crystalline apatite.     

Increased osteoblast adhesion on nanoparticulate crystalline hydroxyapatite functionalized with KRSR

The present in vitro study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate for novel orthopedic applications. Specifically, nano-crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by hydrothermal treatment for 2 hours at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of control conventional HA particles (or those currently used in orthopedics with micron diameters), the aforementioned calcium phosphate particles were pressed into compacts and sintered at 1100 degrees C for 2 hours. All calcium phosphate-based particles were fully characterized. Results showed that although there was an initial weight gain for all the compacts studied in this experiment, higher eventual degradation rates up to 3 weeks were observed for nano-amorphous calcium phosphate compared with nano-crystalline HA which was higher than conventional HA. Peptide functionalization (with the cell adhesive peptide lysine-arginine-serine-arginine [KRSR] and the non-cell-adhesive peptide lysine-serine-arginine-arginine [KSRR]) was accomplished by means of a three-step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The peptide functionalization was fully characterized. Results demonstrated increased osteoblast (bone-forming cell) adhesion on non-functionalized and functionalized nano-crystalline HA compacts compared with nano amorphous calcium phosphate compacts; both increased osteoblast adhesion compared with conventional HA. To further exemplify the novel properties of nano crystalline HA, results also showed similar osteoblast adhesion between non-functionalized nano crystalline HA and KRSR functionalized conventional HA. Thus, results provided evidence that nanocrystalline HA should be further studied for orthopedic applications.     

Thermal conversion of octacalcium phosphate into hydroxyapatite

The thermal conversion of octacalcium phosphate into hydroxyapatite has been investigated by a crystallographic, thermogravimetric, and calorimetric study. The conversion of octacalcium phosphate takes place through the remotion of three of its five water molecules and yields a poor crystalline apatitic phase. The three water molecules are lost in two steps. The first one, which is reversible, corresponds to the remotion of one water molecule and induces a slight contraction of the unit cell of OCP. The successive remotion of two water molecules, which provokes the structural conversion of OCP into apatite, is in irreversible process. The mechanism of the water loss of OCP is explained in terms of its crystal structure.     

Molecular Modelling of a Multiphosphorylated Sequence Motif Bound to Hydroxyapatite Surfaces

Proteins and peptides containing the multiphosphorylated motif -Ser(P)-Ser(P)- Ser(P)--Glu-Glu- stabilise amorphous calcium phosphate (ACP) in body fluids and bind with high affinity to crystalline calcium phosphate phases such as hydroxyapatite (HA) regulating crystal growth. Binding of this motif to hydroxyapatite surfaces was investigated in this study using molecular modelling techniques. Using a three-step computational procedure, we have determined the relative binding energies of the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu to different crystalline surfaces of HA. This analysis revealed preferences of the motif for (100) and (010) surfaces of the crystal and preferences for particular orientations on a given surface. These preferences are principally governed by electrostatic interactions between the crystal lattice and the peptide with the most stable conformers adopting structures where alternate residues exhibit backbone angles characteristic of a -strand and values of an -helix or a distorted -helix, allowing maximal interaction between the acidic side groups and surface calciums. The results of this study are consistent with experimentally-derived data on the interaction of multiphosphorylated proteins/peptides with HA and have implications for the role of these proteins/peptides in calcium phosphate stabilisation and biomineralisation processes.Electronic Supplementary Material available.