Octacalcium Phosphate Crystals Research Articles

Comparative analysis of bovine serum albumin adsorption onto octacalcium phosphate crystals prepared using different methods.

This study compared bovine serum albumin (BSA) adsorption onto octacalcium phosphate (OCP) materials prepared from two wet preparations in the absence (w-OCP) and presence (c-OCP) of gelatin. Raman spectroscopy was used to analyze the BSA adsorption onto OCPs in a 150 mM Tris-HCl buffer containing 0.5 mM calcium and inorganic phosphate (Pi) ions at pH 7.4 and at 37°C. The degree of supersaturation of the supernatants after the adsorption was determined by measuring the ion composition. The results showed that BSA adsorption onto w-OCP was higher than that for c-OCP. The calcium ion concentration of the supernatant decreased for both w-OCP and c-OCP, whereas the Pi ion concentration increased, approaching OCP equilibria at different saturation levels. BSA adsorbed even onto c-OCP, which included a small amount of gelatin during c-OCP preparation. These results indicate that the biodegradability of w-OCP and c-OCP may be modulated through interactions with serum proteins.

Effect of Surrounding Chemical Environment on Adsorption and Accumulation of Serum Protein onto Octacalcium Phosphate Crystals

Effect of Surrounding Chemical Environment on Adsorption and Accumulation of Serum Protein onto Octacalcium Phosphate Crystals

Effect of pH of Coating Solution on Adhesion Strength of Hydroxyapatite and Octacalcium Phosphate Coatings on AZ31 Magnesium Alloy

Hydroxyapatite (HAp) and octacalcium phosphate (OCP) coatings were formed on a Mg-3Al-Zn (AZ31) alloy with a chemical solution deposition method using a Ca-EDTA solution at various pH levels. The adhesion strength of the coatings was examined using the pull-off method. The microstructures of HAp and OCP coatings were measured X-ray diffraction (XRD). The morphology and composition of the surface and cross section of the samples before and after the adhesion test were characterized using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS), and a 3D profilometer. The results showed that plate-like OCP crystals grew from a continuous OCP layer on the surface of the AZ31 substrate in the case of a pH 6.3 coating solution. At pH values of 7.5 and 8.6, the HAp coating showed a two-layer structure with a HAp rod-like outer layer and a HAp continuous inner layer. Regardless of the pH of the coating solutions, a very thin Mg (OH)2 intermediate layer was formed between OCP or HAp coating and substrate. The highest adhesion strength of the coatings was 6.7±0.5 MPa, achieved at a coating solution pH value of 7.5. A part of Mg (OH)2 layer remained on the substrate, indicating that the delamination occurred in the Mg (OH)2 intermediate layer. The primary particles in the inner layer formed at pH 7.5 was smaller than those at pH 8.6. This result indicates that the initial corrosion of substrate AZ31 at pH 7.5 was more rapidly than that at pH 8.6, presumably leading to the formation of mixed layer of Mg (OH)2 and calcium phosphate. Further investigation is necessary to understand the better adhesion strength at pH 7.5 than that at pH 8.6. This good adhesion could be due to the flawless and rod-like uniform crystal, which had the densest and finest structure on the surface.

Tuning the c-Axis Orientation of Calcium Phosphate Hybrid Thin Films Using Polymer Templates

The orientation of the c-axis in octacalcium phosphate (OCP) nanocrystals that were incorporated into hybrid thin films was successfully tuned using poly(vinyl alcohol) (PVA) thin-film templates of varying thicknesses. This approach was inspired by biomineralization. Thicker PVA templates enhanced the c-axis orientation of the OCP crystals perpendicular to the substrate. Using this approach with a 900 nm thick PVA template, OCP/PVA hybrid thin films (1.8 μm thick) with a c-axis orientation perpendicular to the substrate were formed. Hydroxyapatite (HAP) hybrid thin films that also exhibited a perpendicular c-axis orientation were obtained through the topotactic transformation of the OCP/PVA hybrid thin films in aqueous solution. The thickness change of the polymer templates had a significant effect on the structure of the OCP nanocrystals in the hybrid thin films. The structural control of the OCP hybrid thin films that were formed through the biomineralization-inspired approach allowed the formation of HAP hybrid thin films with controlled structures.

In situ silk fibroin-mediated crystal formation of octacalcium phosphate and its application in bone repair

The development of an ideal scaffold material is critical for the repair of bone defects. Being an important precursor of the mineralized matrix of bone tissue, octacalcium phosphate (OCP) has been considered a promising bone substitute. However, its application is largely limited due to the thermodynamical instability and poor processability of it. In this study, OCP was prepared by co-precipitation in the presence of small amount of silk fibroin (SF), which regulated the crystallization of OCP and led to the formation of SF-OCP complex. The diameters of OCP crystals in OCP, 0.1SF-OCP, 0.3SF-OCP and 1SF-OCP complexes were 489.0 ± 399.1 nm, 102.2 ± 50.7 nm, 94.7 ± 48.4 nm and 223.7 ± 167.6 nm, respectively. However, the shape of OCP crystals did not apparently change by the presence of SF. Further, porous SF/OCP composite scaffolds with pore size of 111.9 ± 33.1 μm were prepared, in which small crystals of SF-OCP complex were embedded in a SF matrix. MC3T3-E1 cells could attach and proliferate well on both the rugged surfaces and the pores of SF/OCP scaffolds, indicating their decent biocompatibility. Further, SF/OCP scaffolds markedly promoted bone regeneration in a rat calvarial critical-sized defect model. Both micro-CT and H&E characterizations showed that bone formation not only occurred around the scaffolds, but also penetrated into their center. Therefore, such SF/OCP composite scaffolds may have potential applications in bone tissue engineering.

Sodium Induces Octacalcium Phosphate Formation and Enhances Its Layer Structure by Affecting the Hydrous Layer Phosphate

Octacalcium phosphate (OCP), a layered calcium phosphate compound, has attracted attention in areas such as biomaterial and pharmacy, as well as the environmental industry, because of its excellent biocompatibility and low environmental load. However, little is known about the effect of alkaline metal ions on OCP formation, even though the Na ion is always present as a pH neutralizing or ionic strength-adjusting agent. Therefore, in this study, we investigated the role of the Na ion in OCP formation from dicalcium phosphate dihydrate through hydrolysis by using solutions with various Na concentrations. When the Na concentration in the treatment solutions increased, the formation of hydroxyapatite as a residual material was inhibited and the purity of OCP was increased. Furthermore, at higher concentrations, OCP crystals evolved layer structures because Na affects the P5 PO4 in the OCP lattice and enhances the HPO4-OH layer structure, which significantly contributes to OCP crystallinity and crystal structu...

Roles of Fluoride on Octacalcium Phosphate and Apatite Formation on Amorphous Calcium Phosphate Substrate

As fluoride is known to affect the biomineralization of calcium phosphates, we examined how the growth at 37 °C of octacalcium phosphate (OCP) and hydroxyapatite (HAp) on an amorphous calcium phosphate substrate is affected by low-dose fluoride (0–2 ppm) in metastable calcium phosphate solution. In the absence of fluoride, highly oriented plate-like OCP crystals grow on the substrate. With 0.7–0.8 ppm fluoride, OCP crystal formation was greatly modulated (the crystals were smaller and their orientation was lower); moreover, tiny plates and needles appeared on the OCP plates. There were two critical fluoride concentrations: (1) at 0.9 ppm, the OCP plates were completely replaced by HAp nanorods; (2) at 1 ppm, the structure of the HAp nanorod assembly changed from an open one to a tightly assembled one. The fluoride suppressed OCP formation, induced HAp nanorod formation, and promoted tight nanorod assembly. The relationship between the fluoride concentration and the formation of OCP and HAp provides insigh...

In vitro model of potential metal cation exchanges in biological apatite.

Biological apatite is ion-doped and provides an active pool for the exchange with foreign impurity ions. In this work, an in vitro model of hydrated metastable octacalcium phosphate (OCP) crystals was established to mimetically investigate the distinct exchange of trivalent and divalent cations (Fe3+ and Sr2+) with biological apatite. Fe3+ significantly promoted the collapses of OCP crystals and the formation of amorphous sol-like ferric phosphates, while Sr2+ facilitated the epitaxial growth and stability of OCP crystals. The involvement of Ca2+ maintained the crystalline integrity and inhibited the ion exchange within OCP crystals. This in vitro model would lay the foundation for the further investigation of the metabolism of biological apatite.

Effect of Octacalcium Phosphate-Based Bone Substitute Materials on Oriented Bone Regeneration

The characteristics and the biological responses of octacalcium phosphate (OCP) crystals, obtained in the presence of natural polymers, were summarized based on our studies reported. OCP obtained with collagen molecules in the solution had a plate-like morphology while OCP obtained with gelatin molecules in the solution exhibited elongated morphology toward long axis of the crystals. Oriented bone matrix formation was observed by the OCP inclusion in gelatin sponge in a critical-sized rat calvaria defect within the implantation periods around 8 weeks. It seems likely that specific crystal property of OCP obtained in distinct preparation conditions may affect bone tissue response probably through the modulation of OCP crystal characteristics.

Enhanced hydrated properties of α-tricalcium phosphate bone cement mediated by loading magnesium substituted octacalcium phosphate

A novel Mg-containing α-tricalcium phosphate (α-TCP) bone cement with excellent wetting compressive strength and suitable setting time has been developed in this work. Mg-substituted octacalcium phosphate (Mg-OCP) crystals with the special morphology was prepared by homogeneous precipitation method, and used to modify the α-TCP bone cement. Mg2+ was successfully introduced into OCP structure, and the filaments/sheet-like morphologies of OCP were obtained by changing the Mg2+ concentration. As added in the α-TCP cement matrix, the effects of Mg-OCP on the wetting compressive strength, setting time and microstructure of the cement system were studied. The addition of Mg-OCP shortened the setting time of cement pastes with the minimum setting time of 15 min, and enhanced the wetting compressive strength of the hydrated cement products with the highest wetting compressive strength of 36.86 MPa. This work highlights the special morphologies of Mg-OCP induced by Mg2+ substitution, which made an effect on the hydration reaction of α-TCP cement; Furthermore, Mg-OCP was supposed to improve the condensation capacities and mechanical properties of α-TCP cement system as a novel cement admixture.

Periodic Surface-Ring Pattern Formation for Hydroxyapatite Thin Films Formed by Biomineralization-Inspired Processes.

Surface morphology is a key factor that might significantly influence the properties of biomaterials. In this study, periodic surface-ring structures have been constructed for calcium phosphate thin films via biomineralization-inspired crystallization process. The patterned octacalcium phosphate crystals have been obtained on poly(2-hydroxyethyl methacrylate) (PHEMA) matrix in the presence of poly(acrylic acid) (PAA). The patterned surface morphologies of the crystal thin films could be tuned by the amount of PAA additives. In addition, the rapid and topotactic transformation to hydroxyapatite (HAP) thin films with surface-ring structures has also been achieved. This study may provide new strategy toward the design of functional calcium phosphate-based thin-film hybrids.

Effect of Mg2+ on acidic calcium phosphate phases grown by electrodeposition

In this work, the effect of Mg2+ ions on the electrodeposition of dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and calcium-deficient hydroxyapatite (CDHA) crystals on a commercially pure titanium (cp-Ti) substrate was evaluated. We demonstrated that Mg2+ ions could change the morphology of the coatings by inhibiting the growth rate of the OCP and CDHA crystals and diminishing the crystallite size of DCPD. The inhibition effect on OCP and CDHA was most likely due to a surface adsorption mechanism since no evidence of a doping process was observed using Rietveld refinement and electron diffraction analyses. Conversely, the presence of Mg2+ ions generated a favorable condition for the nucleation of a new Mg2+-rich DCPD crystal population, presenting smaller crystallite sizes.

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H2O2 generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 μm and rod-like crystals with a diameter of around 0.1 μm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2–5 μm. The spacing between OCP tips of 0.8–1.1 μm was wider than that between HAp tips of 0.2–0.3 μm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy.

Crystallization of Calcium Phosphate in Agar Hydrogels in the Presence of Polyacrylic Acid under Double Diffusion Conditions

We studied the precipitation of calcium phosphate (CaP) in the presence or absence of polyacrylic acid (PAA) under double diffusion conditions in agar hydrogels. Waiting times and first precipitation positions were measured to analyze the CaP crystallization behavior, with the equivalency rule governing the precipitation in gel for a double diffusion process. We speculated that amorphous dicalcium phosphate (ADCP, CaHPO4·nH2O) nucleates at the first precipitation position via the reaction that the Ca2+ ion interacts with the HPO42– ion, subsequently transforming into octacalcium phosphate (OCP) according to the Ostwald rule of stages and finally being converted to the thermodynamically more stable hydroxyapatite (HAp) via dissolution. The irregularly shaped aggregates formed at an early stage of crystallization were a mixture of OCP and HAp. In the later stage, spherulites with a hierarchical structure precipitated, with their inner part composed of OCP crystals and the outer part composed of HAp crystals...

Development of octacalcium phosphate bone substitute material and its elucidation of osteoconductivity mechanism

Event Abstract Back to Event Development of octacalcium phosphate bone substitute material and its elucidation of osteoconductivity mechanism Osamu Suzuki1 1 Tohoku University Graduate of Dentistry, Division of Craniofacial Function Engineering, Japan Osteoconductivity of synthetic octacalcium phosphate (OCP) was found first by its implantation in subperiosteal region of mouse calvaria [1]. New bone appeared on OCP earlier than other calcium phosphate materials, such as synthetic stoichiometric hydroxyapatite (HA), while OCP was converted to apatite structure in situ [1]. Osteoblastic differentiation of mouse bone marrow stromal cells and formation of osteoclasts from mouse bone marrow cells in the presence of osteoblasts were enhanced if they were inoculated on OCP surfaces [2]-[4]. It is becoming clear that the stimulatory capacity of OCP is enhanced during a process of OCP hydrolysis into HA at physiological conditions that induces physicochemical reaction around the OCP crystals [2],[5]-[7]. The stoichiometry and the microstructure of OCP crystals, which could be determined by the preparation conditions, control its osteoconductivity[8]-[10]. Combining OCP with natural polymers, such as collagen, gelatin, alginate and hyaluronic acids, through mixing or co-precipitation processes, improves not only the handling property of OCP but also the bone regenerative capacity as bone substitute materials [11]-[15]. This study was supported in part by Grants-in-aid (17076001, 23106010 and 25670829) from the Ministry of Education, Science, Sports and Culture of Japan (MEXT).

Synthesis and structure of iron- and strontium-substituted octacalcium phosphate: effects of ionic charge and radius.

Octacalcium phosphate (OCP) has received intensive research focus as a main component of bone substitute materials due to its highly osteoconductive and biodegradable characteristics. In this work, OCP was synthesized using chemical precipitation methods. Biologically relevant iron ions (Fe3+) and strontium ions (Sr2+) which have different ionic charges and radii were successfully introduced into OCP crystal structure, and their effects on the formation, phase components and structure of OCPs were investigated. The incorporation of Fe3+ and Sr2+ led to lattice expansion of OCP. Both ionic substitutions had slight effects on the morphology and microstructure of typical plate-like OCP crystals. In particular, nanosized particles containing rich Fe were deposited on the surface of plate-like Fe3+-substituted OCP crystals, which confirmed the influence of iron substitution on the corresponding crystal surface nature. This work highlights the different replacements of complex Ca sites by Fe and Sr in the apatite layers and hydrated layers of OCP crystal structure, which gives more possible accounts for foreign trivalent and divalent cations.

Enhancement of HPO<sub>4</sub>–OH layered structure in octacalcium phosphate and its morphological evolution by acetic acid

We show that acetic acid enhances the HPO4–OH layer structure of octacalcium phosphate (OCP) and hypothesize that the presence of acetic acid is essential for obtaining large-sized highly crystalline OCP with relatively high-temperature (∼65°C) aqueous-solution synthesis methods. The OCP crystal consists of three types of layer structures (apatite-like, transition, and HPO4–OH). X-ray diffraction (XRD) analysis indicates that increasing acetic acid concentration leads to the development of their greater structure. Infrared (IR) and nuclear magnetic resonance (NMR) spectroscopic measurements indicated development of the highly HPO4–OH layer-structure. The enhanced layer structure development caused by acetic acid is in contrast to the generally observed decrease in HPO4–OH layer structure in the presence of –COOH-containing molecules such as citrate and succinate.

Characterization of partially hydrolyzed OCP crystals deposited in a gelatin matrix as a scaffold for bone tissue engineering

The present study was designed to investigate how hydrolysis of octacalcium phosphate (OCP) into hydroxyapatite is affected by the presence of gelatin (Gel) molecules and how osteoblastic cells respond to the resultant OCP hydrolyzate/Gel composites as the hydrolysis advances. OCP was prepared from a solution containing calcium and phosphate ions and Gel molecules, having a composition to produce a 40 wt% OCP as a final co-precipitate as the OCP/Gel. The precipitate was further incubated up to 40 h to advance the hydrolysis of OCP. These precipitates were processed to mold OCP/Gel sponges through lyophilization and dehydrothermal treatment. Chemical analysis, X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and selected area electron diffraction revealed that the hydrolysis of OCP/Gel composite in hot water advanced in a time-dependent manner and faster than hydrolysis of OCP alone. The effect of Gel on the OCP hydrolysis was further examined in the presence of distinct concentrations of Gel molecules in hot water, showing that the Gel enhanced the hydrolysis as the concentration increased. Proliferation and differentiation of mouse bone marrow stromal ST-2 cells on the hydrolyzed OCP/Gel composites were compatible with Gel sponge alone after 21 days of culture, suggesting that these composites could be a candidate as a scaffold in bone tissue engineering.

Induction suspension plasma sprayed biological-like hydroxyapatite coatings.

Substituted hydroxyapatite coatings with different ions (Mg, Na, K, Cl, F) have been developed by the induction suspension plasma spray process. Suspensions were prepared with sol-gel. The main objective of this study was to demonstrate that induction suspension plasma spray technology possesses high material composition flexibility that allows as-sprayed coatings to closely mimic natural bone composition. Long-term invitro behaviour of as-sprayed substituted coatings was evaluated with simulated body fluid. Data on the suspensions showed the formation of a pure hydroxyapatite phase. Transmission electron microscopy characterized various preparation stages of the suspensions. As-sprayed samples were distinguished by X-ray diffraction and scanning electron microscopy. Substituted elements were quantified by neutron activation. A well-crystallized hydroxyapatite phase was produced with concentration in various substitutions very close to natural bone composition. Ca/P and (Ca + Mg + Na + K)/P ratios provided evidence of the introduction of different cations into apatite structures. The immersion of samples into simulated body fluid led to the nucleation and growth of a flake-like octacalcium phosphate crystal layer at the surface of as-sprayed coatings after one week. Proof of octacalcium phosphate transformation and its partial dissolution and direct re-precipitation into apatite was disclosed by local energy dispersive spectroscopy and microstructure observation. Formation of a Ca/P ratio gradient from the precipitated layer surface to the as-sprayed coatings interface was observed after four weeks once the octacalcium phosphate crystals reached a critical size, resulting in the formation of a rich apatite layer at the interface after six weeks. A set of mechanisms has been proposed to explain these findings.

<i>In Situ</i> Atomic Force Microscopy Observation of Octacalcium Phosphate (100) Face Dissolution in Weak Acidic Solutions

Dissolution of the (100) face of octacalcium phosphate (OCP) single crystal in weak acidic solutions (pH = 6.5; 25°C) was observed in situ using atomic force microscopy. Monomolecular steps (2.0 nm high) were observed; they originated from etch pits or crystal edges. Advancement of the dissolution process led to precipitation of nanoparticles as small as ~10 nm even though the solution was undersaturated with respect to OCP. This precipitation of nanoparticles was accompanied by a drastic decrease in the dissolution rate; however, the substrate OCP continued to dissolve, indicating that dissolution and growth occurred simultaneously on the same surface. The precipitated nanoparticles coalesced and eventually covered the entire surface without changing the surface morphology of the substrate crystal. The step height after complete coverage was ~2.0 nm, the same as that observed on the dissolving OCP surface. These findings indicate that the precipitated phase was a pseudomorph of OCP crystal.