Single Crystal Hydroxyapatite Research Articles

Synthesis of plate-like single-crystal hydroxyapatite rods with c-axis orientation by biotemplate small intestinal submucosa

Abstract Since hydroxyapatite crystals are characteristically c-axis orientation on the surface of vertebrate long bone, c-axis orientation HAP biomimetic synthesized will find extensive applications in long bone growth, remodeling and fracture healing. In this paper, plate-like single-crystal HAP rods with c-axis orientation was successfully synthesized at bone mineralization conditions in vivo, with small intestinal submucosa membrane as biomineralization template. The samples were characterized by XRD, FIIR, SEM, TEM and EDS to unveil the phase structure, composition, morphology, and a plausible growth mechanism was proposed. The results showed that morphology of samples changed from flower-like to plate-like with extension reaction time from 1 day to 10 days. The plate-like HAp rods were single-crystal with c-axis orientation. A unit of plate-like HAP rods is about 70 µm and the width is 4 µm. Phase composition transformed from octocalcium phosphate and HAp biphase to HAp phase with very little octocalcium phosphate phase. Finally, biocompatibility of the samples was evaluated by CCK8. The samples without significant cytotoxicity conformed to the need for substitute materials of bone regeneration.

Hierarchy effect on electronic structure and core-to-valence transitions in bone tissue: perspectives in medical nanodiagnostics of mineralized bone

Electronic structure and core-to-valence transitions in bone tissue are examined in the framework of the morphological 3DSL model that takes into account (i) structural and functional organization of the skeleton in the normal and pathological conditions and (ii) peculiarities of electron wave propagation in a three-dimensional superlattice of “black-nanocrystallites-in-muddy-waters”. Our focus is on the HAP-to-bone red shifts of core-to-valence transitions near Ca and P 2p and O 1s edges in single-crystal hydroxyapatite (HAP) Ca10(PO4)6(OH)2. The origin of the HAP-to-bone shift is discussed and the extended comparative analysis of the experimental data is performed. The detected spectral shift is assigned with the effect of hierarchical organization of bone tissue. This hierarchy effect on the core-to-valence transition energies is regarded as a promising tool for medical imaging and perspective pathway for nanodiagnostics of mineralized bone.

Biomineralization of nanoscale single crystal hydroxyapatite.

The chemical and physical characteristics of nanocrystalline hydroxyapatite particles which formed during the subcutaneous implantation of crab shell in Sprague-Dawley rats were studied using selected area electron diffraction (SAED) and high resolution transmission electron microscopy (HRTEM). The initial SAED characterization evidence indicated the presence of an amorphous calcium phosphate phase. The electron dense nanophase particles which formed in the wound healing zone displayed broad diffuse rings which usually indicate a low crystalline order or amorphous phase. High resolution transmission electron microscopy (HRTEM) revealed that these mineralized regions contained discrete single crystal particles less than 5nm in size. Micrographs taken at successively higher magnifications revealed very small nanoparticles with a hexagonal arrangement of ion channels with characteristic spacing of 0.54nm and 0.23nm. This study revealed that single crystal hydroxyapatite nanoparticles consisting of only a few unit cells formed via a biomineralization directed process.

Vibrational algorithms for quantitative crystallographic analyses of hydroxyapatite-based biomaterials: I, theoretical foundations.

The Raman spectroscopic method has quantitatively been applied to the analysis of local crystallographic orientation in both single-crystal hydroxyapatite and human teeth. Raman selection rules for all the vibrational modes of the hexagonal structure were expanded into explicit functions of Euler angles in space and six Raman tensor elements (RTE). A theoretical treatment has also been put forward according to the orientation distribution function (ODF) formalism, which allows one to resolve the statistical orientation patterns of the nm-sized hydroxyapatite crystallite comprised in the Raman microprobe. Close-form solutions could be obtained for the Euler angles and their statistical distributions resolved with respect to the direction of the average texture axis. Polarized Raman spectra from single-crystalline hydroxyapatite and textured polycrystalline (teeth enamel) samples were compared, and a validation of the proposed Raman method could be obtained through confirming the agreement between RTE values obtained from different samples.

Spatial Organization of Hydroxyapatite Nanorods on a Substrate via a Biomimetic Approach

The growth of uniform single-crystal HAp (hydroxyapatite) nanorods on specific sites on a substrate is achieved. HAp nanorods were synthesized under hydrothermal conditions in a solution containing calcium and phosphate ions, urea as a source of base, and with gelatin as a structure directing agent. The nanorods are hundreds of micrometers in length and 50–200 nm in width. Without gelatin, platelike HAp crystals were formed. To grow these uniform HAp nanorods on specific sites on a substrate, the surface of a gold-coated glass slide was coated by a self-assembled monolayer of 11-mercaptoundecanoic acid by stamping with a patterned PDMS (polydimethylsiloxane) stamp. The HAp nanorods were grown exclusively in the carboxylate functionalized regions. Selective area electron diffraction analyses show that the single-crystal HAp nanorods formed on the substrate grow along the crystallographic c-axis. In comparison to the nonpatterned sample, an increase of the X-ray diffraction intensity ratio of (00l)/(h00) peaks in the patterned sample indicates that nanorods are preferentially oriented with their c-axes perpendicular to the substrate, which was confirmed by SEM cross-sectional images. In this strategy, both the morphology and the deposition sites of HAp nanorods are controlled.

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.

Hydroxyapatite supported cobalt catalysts for hydrogen generation

The controlled generation of H2 from storage materials by using an efficient catalytic support is a highly sought after technology; however, the majority of successes utilize expensive materials considered unfeasible. In our report on the creation of a novel, durable, and inexpensive catalytic support material for hydrogen generation, we examine a critical surface modification of hydroxyapatite (HAP) with cobalt ions to provide the necessary catalytic transition metal for the fast hydrolysis of the hydrogen storage material, sodium borohydride (NaBH4). By altering the morphology and composition of the HAP crystal supports, we revealed novel methods for enhancing the hydrogen generation rates. Particularly, lowering the Ca composition during synthesis of the HAP crystals afforded a Ca deficient HAP capable of exhibiting a higher surface coverage of cobalt, thereby eliciting faster hydrolysis reaction rates in comparison with the amorphous HAP control having the characteristic Ca content for HAP. A more significant increase in hydrogen generation was observed when using single crystal HAP in comparison with amorphous and calcium deficient HAP supports. Despite the smaller surface area of the hydrothermally prepared single crystal HAP, it provided significantly faster hydrogen generation. Each of the HAP supports exhibit repeatability with catalytic efficiency decreasing by approximately 25% over 3weeks upon repeated daily exposure to solutions of the hydrogen storage material NaBH4. Through these experiments, we proved that altering the composition and morphology of cobalt ion exchanged HAP supports can offers a useful means for increasing the rate of controlled hydrogen generation.

Evolutionary Screening of Collagen-like Peptides That Nucleate Hydroxyapatite Crystals

The biogenesis of inorganic/organic composite materials such as bone typically involves the process of templated mineralization. Biomimetic synthesis of bone-like materials therefore requires the development of organic scaffolds that mediate mineralization of hydroxyapatite (HAP), the major inorganic component of bone. Using phage display, we identified a 12-residue peptide that bound to single-crystal HAP and templated the nucleation and growth of crystalline HAP mineral in a sequence- and composition-dependent manner. The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp) of type I collagen, a major component of bone extracellular matrix. Using a panel of synthetic peptides, we defined the structural features required for mineralizing activity. The results support a model for the cooperative noncovalent interaction of the peptide with HAP and suggest that native collagen may have a mineral-templating function in vivo. We expect this short HAP-binding peptide to be useful in the synthesis of three-dimensional bone-like materials.

Milling media effects on synthesis, morphology and structural characteristics of single crystal hydroxyapatite nanoparticles

This paper presents a dry mechanochemical process to produce hydroxyapatite (HAp) nanoparticles. Two distinct chemical reactions are introduced to prepare HAp powders using milling process. Structural and morphological properties of the obtained materials are studied by X-ray diffraction and transmission electron microscopy. The results reveal that the single crystal HAp nanoparticles have been successfully produced in metallic and polymeric vials through two different experimental procedures. Tempered chrome steel and polyamide-6 materials are adopted as the metallic and polymeric vials respectively. Nanoellipse, nanorod and nanosphere powders are obtained in these experimental procedures. Moreover, the obtained HAp powders through two distinct reactions exhibit average sizes about 12 and 15 nm using the tempered chrome steel vials, and about 16 and 17 nm using the polyamide-6 vials. The results indicate that single crystal HAp nanoparticles produced in polyamide-6 vials have suitable morphology and high production efficiency without any chemically stable contaminations.

Effect of Salinity on Hydroxyapatite Dissolution Studied by Atomic Force Microscopy

The complexity of bone tissue and the lack of techniques for directly probing bone surfaces in vivo have hindered studies on the fundamental mechanisms of bone mineral remodeling. Here, we addressed these issues by using single crystal hydroxyapatite (HAP) as a well-defined bone surface model and directly observe its surface using in situ atomic force microscopy. Specifically, we investigated the effects of NaCl concentration on the dissolution of HAP (100) surfaces and found that NaCl strongly suppressed HAP dissolution kinetics, including at physiological ion concentrations. These findings indicate that local ion concentrations in vivo may contribute significantly to the stability of bone mineral. Moreover, our analysis of HAP surfaces over a broad range of pH conditions suggests that only one kind of surface termination exists exclusively in the solutions we used. This observation is important for understanding the surface chemistry of HAP. Our molecular level, real-time observations of HAP dissolution...

Micromechanical properties of single crystal hydroxyapatite by nanoindentation

Knowledge of the intrinsic properties of hydroxyapatite (HAp) single crystals is important for the design of natural systems and will assist further improvements of manufactured biomaterials. Nanoindentation provides a useful tool for determining mechanical properties such as the hardness, elastic modulus and fracture toughness of small samples. A Berkovich indenter was placed on the side and basal planes of a natural single crystal of Durango HAp. The hardness and elastic modulus values obtained revealed higher values for the base (7.1 and 150.4 GPa) compared to the side (6.4 and 143.6 GPa). The cracking threshold, i.e., the load at which cracking initiates, revealed earlier crack formation on the base (at 8 mN) compared to the side (at 11 mN). Fracture toughness was measured as 0.45 ± 0.09 and 0.35 ± 0.06 MPa m 1/2 for the side and basal plane, respectively. These results suggest that crystals are less prone to cracking and resist microcrack events better on the side, which is useful in bone, while exposing the base, the hardest face, to minimize mass loss from abrasion with teeth.

Synthesis of Hydroxyapatite Nanoplates by the Hydrothermal Reaction Method with Anionic Amino Acid Surfactant

By hydrothermal reaction method with the control of anionic amino acid surfactant N-lauroylsarcosine sodium(Sar-Na),single crystal hydroxyapatite(HAP) nanoplates were synthesized and different synthetic conditions were carefully studied.The products were characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray diffraction(XRD),and Fourier transform infrared(FTIR) spectroscopy.Results showed that hydroxyapatite nanocrystals with lengths of 0.5-1.0 μm,a width of 30 nm,and a high aspect ratio(20:1) were obtained.By increasing the concentration of N-lauroylsarcosine sodium,the morphology of the hydroxyapatite varied from oval to plate-like.These results show that N-lauroylsarcosine sodium can induce the formation of hydroxyapatite with different morphologies.The current work provides a new approach to tune the aspect ratio of hydroxyapatite nanoplates.

Synthesis of nanosize single-crystal hydroxyapatite via mechanochemical method

Single-crystal hydroxyapatite (HAp) nanorods and nanogranules have been synthesized successfully by a mechanochemical process using two distinct experimental procedures. The experimental outcomes are characterized by transmission electron microscopy (TEM), and powder X-ray diffraction (XRD) techniques. In this work, the feasibility of using polymeric milling media to prepare hydroxyapatite nanoparticles is described. The resulting hydroxyapatite powder exhibits an average size of about 20 to 23 nm. Final results indicate that the proposed synthesis strategy provides a facile pathway to obtain single-crystal HAp with high quality and suitable morphology.

Defect Induced Asymmetric Pit Formation on Hydroxyapatite

Defect sites on bone minerals play a critical role in bone remodeling processes. We investigated single crystal hydroxyapatite (100) surfaces bearing crystal defects under acidic dissolution conditions using real-time in situ atomic force microscopy. At defect sites, surface structure-dependent asymmetric hexagonal etch pits were formed, which dominated the overall dissolution rate. Meanwhile, dissolution from the flat terraces proceeded by stochastic formation of flat bottom etch pits. The resulting pit shapes were intrinsically dictated by the HAP crystal structure. Computational modeling also predicted different step energies associated with different facets of the asymmetric etch pits. Our microscopic observations of HAP dissolution are significant for understanding the effects of local surface structure on the bone mineral remodeling process and provide useful insights for the design of novel therapies for treating osteoporosis and dental caries.

Biomimetic Mineralization Induced by Fibrils of Polymers Derived from a Bile Acid

A polymer containing bile acid pendant groups was prepared by free-radical polymerization of the 3alpha-methacrylate derivative of cholic acid. The polymer formed fibrils of about 1 nm in diameter in aqueous media and further assembled into bundles or lamella plates, as observed by transmission electron microscopy. After immersing the fibrils in simulated body fluid with ion concentrations equivalent to those in human plasma, plate-like crystals formed on the surface of the fibril assembly. The diffraction pattern corresponding to the (001) plane of hydroxyapatite was identified by transmission electron microscopy and selected area electron diffraction. The plate-like, single crystal hydroxyapatite formed on such a polymeric assembly may be useful in the matrix design for biomimetic mineralization and in the development of scaffold materials for hard tissue engineering.

Hydrothermal microemulsion synthesis of stoichiometric single crystal hydroxyapatite nanorods with mono-dispersion and narrow-size distribution

The stoichiometric single crystal hydroxyapatite (HAp) nanorods with mono-dispersion and narrow-size distribution in diameter were successfully synthesized via a hydrothermal microemulsion method, and the as-synthesized powders were characterized by XRD, FETEM, FTIR and ICP-AES. In the present work, a novel technique of w/o microemulsions of CTAB/ n-pentanol/ n-hexane/water under hydrothermal condition to synthesize single crystal HAp nanorods with diameter 25–40 nm and length 55–350 nm, was described. The homogeneity in size distribution and shape of the HAp nanorods was probably attributed to the w/o nano-reactors and the soft template of the surfactants, and the high crystallization of the products was attributed to the hydrothermal treatment. The sintering ability of the nanorods and the mechanical properties of the fabricated HAp bioceramics were also preliminarily investigated. The results showed that the fabricated HAp bioceramics possessed high mechanical properties.

Synthesis of single-crystal HAP nanorods

Single-crystal hydroxyapatite (HAP) nanorods have been successfully synthesized by the reverse microemulsion method, and characterized by high-resolution transmission electron microscope (HRTEM), infrared spectra (IR), and powder X-ray diffraction (XRD). In this work, the feasibility of using quaternary reverse microemulsion (TX-100 + CTAB/ n-butanol + n-hexanol/cyclohexane/water) to prepare hydroxyapatite nanorods with diameter 8–15 nm and length 25–50 nm, is described. The homogeneity in size and shape of hydroxyapatite nanoparticles observed by TEM is probably attributed to the different stabilization functions of different kinds of surfactant on the interfacial film.

TEM examination of single crystal hydroxyapatite diffraction

TEM examination of single crystal hydroxyapatite diffraction

Mechanical Properties of Hydroxyapatite and OH-carbonated Hydroxyapatite Single Crystals

Single-crystal hydroxyapatite and OH-carbonated hydroxyapatite have bending strength much higher than that of dense hydroxyapatite ceramic, indicating potential applicability to a load-bearing biomaterial. However, the effects of carbonate on the strength are less clear. The objective of the present study was to determine the bending strength and Young's modulus of these single crystals with carbonate contents from 0 to 0.62 in CO2 wt%. Three-point bending tests were performed by means of a modified ultra-microhardness tester with a span of 380 microm and a bending direction <210> of the single crystals. The crystals were broken in air, water, and air after immersion in a cell culture medium for 3 wks. The average Young's modulus of the single crystals was from 54 to 79 GPa. The average bending strength of the single crystals in air was 500+/-184, 468+/-205, 513+/-151, and 450+/-162 MPa for those with 0, 0.09, 0.37, and 0.62 wt% carbonate, respectively. No significant decrease in strength was found for hydroxyapatite single crystals both in water and after the immersion in the medium. However, the strength of OH-carbonated hydroxyapatite single crystals decreased significantly by 23 to 43% in water in proportion to the carbonate content. The strength of single crystals with 0.37 and 0.62 wt% carbonate decreased significantly, even after the immersion in the medium. Therefore, hydroxyapatite single crystals are superior to OH-carbonated hydroxyapatite single crystals as a biomaterial for a load-bearing purpose.

破骨細胞性骨吸収における骨無機塩および有機基質の役割

The aim of this study is to investigate the properties of bone substrates which are responsible for induction of bone-resorptive behavior of osteoclasts. For this purpose, the recently developed culture system in which the bone-resorptive activity of isolated osteoclasts can be analized on various natural or artificial substrates has been utilized.The results are follows:1. Resorption pits by isolated osteoclasts occured on bovine enamel, bovine dentin, egg shell, clam shell and lingula shell, containing hydroxyapatite, calcite and aragonite respectively.2. The resorptive activity of isolated osteoclasts was significantry lower on demineralized bone slices than that on untreated bone.3. Extracellular calcium from 2mM to 3mM stimulated resorptive activity although 6mM or the above inhibited the activity in 15h of culture.4. Although no resorption was seen on the surfaces of polycrystalline synthetic hydroxyapatite, geological single crystal hydroxyapatite and α-tricalcium phosphate, many pits were observed on octacalcium phosphate.5. Resorption pits were created on the bone slices made anorganic by hydrazine treatment or sintering at 700°C and 900°C.6. Electron microscopy revealed that elaborated ruffled border and clear zone developed in the osteoclasts on the mineralized bone, on the other hand, irregularly arranged ruffled border and poorly-organized appeared in the osteoclasts on the demineralzed bone.These observations have suggested that bone mineral is responsible for induction of bone resorption by osteoclasts in close relation to development of ruffled border in osteoclasts.