Plate-like Hydroxyapatite Research Articles

C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization

Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration.

Synthesis and ultrastructure of plate-like apatite single crystals as a model for tooth enamel

Hydroxyapatite (HAp) is an inorganic constituent compound of human bones and teeth, with superior biocompatibility and bioactivity characteristics. Its crystal structure is hexagonal, characterized by a(b)- and c-planes. In vertebrate long bones, HAp crystals have a c-axis orientation, while in tooth enamel, they have an a(b)-axis orientation. Many methods can be used to synthesize c-axis oriented HAp single crystals; however, to the best of our knowledge, there have been no reports on a synthesis method for a(b)-axis oriented HAp single crystals. In this study, we successfully synthesized plate-like HAp crystals at the air–liquid interface of a starting solution via an enzyme reaction of urea with urease. Crystal phase analysis and ultrastructure observations were carried out, and the results indicated that the particles were single crystals, with almost the same a(b)-axis orientation as tooth enamel. It is hoped that by utilizing their unique surface charge and atomic arrangement, the resulting particles can be used as a high-performance biomaterial, capable of adsorbing bio-related substances and a model for tooth enamel.

Protein adsorption on single-crystal hydroxyapatite particles with preferred orientation to a(b)- and c-axes

Adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) proteins with preferred orientation to a(b)- and c-axes of single-crystal hydroxyapatite (HAp) particles, was investigated. Fiber-like HAp single crystal particles (aHAp) and plate-like HAp single crystal particles (cHAp) were used as models for a(b)-plane and c-plane oriented HAps, respectively, together with randomly shaped HAp particles (iHAp) as a control. The selective adsorption behaviors of negatively charged BSA and positively charged LSZ on these HAp particles were examined in a phosphate buffered saline at pH 7.3 and 25°C for 48h. The amount of BSA adsorption, normalized for specific surface area, was in the order of aHAp>iHAp>cHAp; however, the order for LSZ was reversed as cHAp>iHAp ≒ aHAp. These results indicate that the a(b)- and c-planes of HAp crystal have high specificity for the adsorption of acidic or basic proteins.

Mineralization of hydroxyapatite crystallites on zein microspheres

AbstractBioinspired mineralization process has been extended to the formation of bonelike hydroxyapatite (HAp) coatings on biodegradable polymer substrates. HAp‐coated zein microspheres were prepared through phase separation procedure, followed by incubation in modified simulated body fluids (mSBF). The morphologies of mineralized zein microspheres with different diameters and incubation time were investigated by scanning electron microscopy (SEM). The nature of the deposited mineral phase was characterized by energy‐dispersive spectroscopy (EDS) and wide‐angle X‐ray diffraction (XRD). The results showed that as the average diameter of zein microspheres increased to 1.13 μm, randomly oriented minerals were observed on the surface of microspheres. Two characteristic HAp diffraction reflections 002 and 211 centered at 2θ of 25.6° and 32.0° were ascertained on the XRD patterns of the mineralized microspheres. Incubation of zein microspheres with larger diameters in mSBF led to nucleation and growth of minerals on the surface. Even larger number of minerals were deposited and a porous structure of platelike HAp crystals was obtained after incubation for 21 days. The novel zein/HAp microspheres may have a wide variety of potential applications in bone regeneration and tissue engineering. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers

Mineralization regulation and biological influence of bioactive glass-collagen-phosphatidylserine composite scaffolds

Biomimetic scaffolds are appealing products for the repair of bone defects using tissue engineering strategies. In the present study, novel biomimetic composite scaffolds, with similar properties to natural bone, were prepared, blended and cross-linked with bioactive glass, type I collagen and phosphatidylserine. When exposed to cell culture solution in the absence of a cellular source, the composite scaffolds form crystals with octahedral structure. These crystals are similar to the products derived from MC3T3-E1 cell mineralization within the composite scaffolds, with respect to both composition and morphology. Furthermore, crystals with octahedral structure were observed to develop into plate-like hydroxyapatite. The bio-mineralization behavior of the composite scaffolds is likely influenced by inorganic components. Finally, a rabbit tibia defect model shows that the highly bioactive properties of the investigated composites result in excellent bone repair.

Controlled synthesis of hydroxyapatite crystals templated by novel surfactants and their enhanced bioactivity

In this paper, we report that hydroxyapatite (HAP) nanocrystals with various shape and size have been successfully prepared by a dual template-assisted hydrothermal synthesis approach in isopropanol/water mixed solvents. Herein, we chose 4-aminobenzenesulfonic acid (ABSA) and polyethylene glycol 4000 (PEG-4000) as structure-directed templates, respectively. By gradually changing the mass ratios (R) of PEG-4000 to ABSA, the obtained HAP samples can undergo distinct morphological evolution accordingly. A class of unique sheet-like HAP sample could be formed when only using ABSA as template. Moreover, when the R value was changed to 1/4, uniform and high-yielding HAP plate-shaped structure can be obtained. When only using PEG-4000 as a template, almost novel and wedge-shaped HAP samples can be obtained. The results demonstrated that the obtained plate-like HAP sample has single-crystalline behavior and uniform distribution in both size and shape. Additionally, we perform and evaluate the bioactivity of HAP in simulated body fluid (SBF), indicating that the two obtained HAP samples can possess apparently improved bioactivity compared to previous literature reports, and bone-like apatite could be readily formed on the HAP surface through different soaking periods in SBF. Notably, for the first time, a novel method for evaluating the in vitro bioactivity of HAP has been proposed. Therefore, the presented synthetic route for HAP is well-controlled and facilitated, which could be extended to the preparation of other biomaterials with specific morphologies and architectures.

Plate-like hydroxyapatite nanoparticles synthesized by the hydrothermal method

In this article, calcium nitrate (Ca(NO 3) 2) and disodium hydrogen phosphate (Na 2HPO 4) are used as calcium and phosphorous sources to prepare hydroxyapatite nanoparticles by the hydrothermal method. Plate-like nanocrystals of hydroxyapatite are synthesized with the aid of sodium tripolyphosphate. The results show that sodium tripolyphosphate increases the diameters of the hydroxyapatite nanoparticles during the hydrothermal process. When the concentration of sodium tripolyphosphate reaches 0.015 M, the average aspect ratio of those nanoparticles is close to 1. The strong surface adsorption caused by sodium tripolyphosphate may answer for the morphological change of hydroxyapatite crystal.

Crystal Structure of Hydroxyapatite Nanorods Synthesized by Sonochemical Homogeneous Precipitation

Using a homogeneous precipitation method in an ultrasound field, we synthesized nanosized, platelike hydroxyapatite (HAp). The internal structure of these platelike formations consists of specifically oriented and laterally connected nanorods. The synthesized HAp nanorods have a length of about 500 nm and a diameter of about 100 nm. A closer inspection of the microstructure of a single nanorod revealed a highly regular and defect-free lattice with unique crystallographic plane orientations. The obtained structure was related to the influence of the ultrasound on the growth mechanism. The samples were characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM).

Freeze casting of porous hydroxyapatite scaffolds. I. Processing and general microstructure

Freeze casting of aqueous suspensions on a cold substrate was investigated as a method for preparing hydroxyapatite (HA) scaffolds with unidirectional porosity. In the present paper, we report on the ability to manipulate the microstructure of freeze-cast constructs by controlling the processing parameters. Constructs prepared from aqueous suspensions (5-20 volume percent particles) on a steel substrate at -20 degrees C had a lamellar-type microstructure, consisting of plate-like HA and unidirectional pores oriented in the direction of freezing. Sintering for 3 h at 1350 degrees C produced constructs with dense HA lamellas, porosity of approximately 50%, and inter-lamellar pore widths of 5-30 microm. The thickness of the HA lamellas decreased but the width of the pores increased with decreasing particle concentration. Decreasing the substrate temperature from -20 degrees C to -196 degrees C produced a finer lamellar microstructure. The use of water-glycerol mixtures (20 wt % glycerol) as the solvent in the suspension resulted in the production of finer pores (1-10 microm) and a larger number of dendritic growth connecting the HA lamellas. On the other hand, the use of water-dioxane mixtures (60 wt % dioxane) produced a cellular-type microstructure with larger pores (90-110 microm). The ability to produce a uniaxial microstructure and its manipulation by controlling the processing parameters indicate the potential of the present freeze casting route for the production of scaffolds for bone tissue engineering applications.

Adsorption Processes of Gly and Glu Amino Acids on Hydroxyapatite Surfaces at the Atomic Level

The regulation mechanism of organic additives on the crystallization of inorganic crystal is fundamentally important in biomineralization. Experimentally, it was found that the amino acids glycine (Gly) and glutamic acid (Glu) could lead to the formation of rod- and plate-like hydroxyapatite (HAP) crystallites, respectively. The detailed adsorption behavior of Gly and Glu on HAP crystal faces was studied by molecular dynamics (MD) simulation. The specific adsorption sites and patterns of Gly and Glu on the (100) and (001) faces of HAP crystals were revealed at the atomic level. The amino acids adsorbed on the HAP (001) and (100) faces with their positive amino groups occupied vacant calcium sites, and their negative carboxylate groups occupied vacant P or OH sites precisely and formed an ordered adsorption layer. The atomic force microscopy pulling simulation and free energy calculation showed that Glu was much more difficult to depart from the HAP (001) face than that from the (100) face. This result indicated that Glu preferred to adsorb strongly onto the HAP (001) face, which resulted in the formation of plate-like HAP. However, Gly did not show any significantly preferential adsorption between these two HAP faces. Thus, the habits of HAP, rod-like crystallites, were not altered during the HAP crystallization in the presence of Gly. Combined with experimental results, our study demonstrated that the MD simulation of interfacial structures could improve our understanding of biological regulation in mineralization processes at the atomic level.

Preparation of hydroxyapatite ceramics by hydrothermal hot-pressing method at 300 °C

Hydroxyapatite (HA) ceramics were prepared by a hydrothermal hot-pressing (HHP) method at a low temperature (300 °C). DCPD (CaHPO4·2H2O) + Ca(OH)2, OCP (Ca8H2(PO4)6·5H2O) + Ca(OH)2, DCPD + NH3·H2O, OCP + NH3·H2O or α-TCP (Ca3(PO4)2) + NH3·H2O were used as the precursors. The mixture was treated by HHP under a condition of 300 °C/40 MPa. In sample DCPD + Ca(OH)2 and OCP + Ca(OH)2, the HA ceramics obtained showed a porous and homogenous microstructure, and the bending strength were 9.9 MPa and 10.9 MPa, respectively. In sample α-TCP+NH3·H2O, rod-like HA crystals produced. When the starting materials were DCPD + NH3·H2O, OCP + NH3·H2O, the HA particles produced exhibited plate-like features. It appeared that the plate-like HA particles stacked into a lamellar structure. The formation of the lamellar structure leads to a noticeable improvement in fracture property of the HA ceramic. The bending strength and the fracture toughness of the sample prepared from OCP and ammonia water reach 90 MPa and 2.3 MPam1/2, respectively.

Synthesis of Hydroxyapatite Crystals Using Amino Acid-Capped Gold Nanoparticles as a Scaffold

Inorganic composites are of special interest for biomedical applications such as in dental and bone implants wherein the ability to modulate the morphology and size of the inorganic crystals is important. One interesting possibility to control the size of inorganic crystals is to grow them on nanoparticles. We report here the use of surface-modified gold nanoparticles as templates for the growth of hydroxyapatite crystals. Crystal growth is promoted by a monolayer of aspartic acid bound to the surface of the gold nanoparticles; the carboxylate ions in aspartic acid are excellent binging sites for Ca(2+) ions. Isothermal titration calorimetry studies of Ca(2+) ion binding with aspartic acid-capped gold nanoparticles indicates that the process is entropically driven and that screening of the negative charge by the metal ions leads to their aggregation. The aggregates of gold nanoparticles are believed to be responsible for assembly of the platelike hydroxyapatite crystals into quasi-spherical superstructures. Control experiments using uncapped gold nanoparticles and pure aspartic acid indicate that the amino acid bound to the nanogold surface plays a key role in inducing and directing hydroxyapatite crystal growth.

Hydroxyapatite coating on titanium by means of thermal substrate method in aqueous solutions

Hydroxyapatite (HAp) films were formed on a titanium substrate in aqueous solutions by the thermal substrate method controlling the substrate temperature and a cathodic electrolysis method supplying hydroxide ions. A local increase in temperature on substrate and the supply of calcium, phosphate, and hydroxide ions near the substrate accelerate the HAp film formation on the substrate in aqueous solutions at ambient temperature and pressure. The HAp can be directly coated only on the substrate quickly by heating the substrate in an aqueous solution. In the cathodic process, reduction of hydrogen peroxide forms hydroxide ions, which results in the formation of a flat, plate-like HAp film.

Morphological stability of hydroxyapatite precursor

Hydroxyapatite powders [Ca10(PO4)6(OH)2] (HAp) were produced via sol–gel method. The thermal crystallization behavior was studied by means of differential thermal analysis, differential scanning calorimetry (DSC), mass spectrometry (MS) and scanning electron microscopy. A variable heating rate approach was used to calculate the activation energy of crystallization of hydroxyapatite from amorphous precursor (235 kJ/mol). The plate-like morphology observed in the hydroxyapatite precursor at 150 °C resulted in plate-like hydroxyapatite.

Synthesis of c Axes Oriented Hydroxyapatite Aggregate

Abstract The c axes oriented hydroxyapatite (HAp) aggregates were synthesized by the hydrolysis of CaHPO4·2H2O (DCPD) at 100 °C in 10 mass% NaOH solution. The HAp was of a plate-like form resembling DCPD. The X-ray diffraction pattern shows that the relative peak intensities of 002 and 004 diffractions increased with the temperature. The crystals in an aggregate were aligned perpendicular to the wide surface of an original plate-like DCPD. HAp crystallites grew in the [00l] direction perpendicular to the wide surface of plate-like HAp aggregate.

Influence of monosaccharides and related molecules on the morphology of hydroxyapatite

The influence of monosaccharides and related molecules on the crystal morphology of hydroxyapatite (HAP) precipitated from chloride-containing supersaturated solutions has been studied by transmission electron microscopy. Pulse addition of aliquots of CaCl 2 to Na 2HPO 4 solutions resulted in the formation of plate-like HAP crystals. In contrast, the presence of the monosaccharides D-glucose, D-galactose, D-mannose, glucuronic acid, N-acetyl glucosamine and D-fructose (phosphate:additive = 6:1) induced the precipitation of needle-shaped HAP crystals elongated along the c-axis. Other molecules, such as sorbitol and 1,2-butanediol, also showed this morphological effect. The order of decreasing aspect ratios was; sorbitol = 1,2-butanediol (25:1)> fructose (20:1)> glucose = galactose = mannose = glucuronic acid = N-acetyl glucosamine (10:1). Expression of the needle-like morphology is discussed in terms of kinetic factors involving nucleation and growth. The former is dependent on the exclusion of Cl - from HAP nuclei due to additive interactions with an amorphous calcium phosphate precursor phase. The latter is related to the strength of additive binding with the subsequent development of {100} HAP crystal faces.

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent.

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.