Improved bioabsorbability of synthetic hydroxyapatite through partial dissolution-precipitation of its surface

Even though synthetic hydroxyapatite (HAp) has a chemical composition similar to the mineral phase of bone, it is minimally absorbed and replaced by bone tissue. This could be because HAp is composed of compactly arranged apatite crystals with homogenously large grains. In this study, the surface and non-stoichiometry of the synthetic HAp crystals was modified by partial dissolution and precipitation (PDP) to improve bioabsorbability of HAp. In vitro cell culture demonstrated that more osteoclasts were activated on PDP-HAp compared with HAp. In vivo implantation using a rabbit bone defect model revealed that PDP-HAp was gradually degraded and was replaced by bone tissue. Consistent with the in vitro results, more osteoclasts were activated in PDP-HAp than in HAp, indicating that the former was absorbed through the stimulation of osteoclastic activity. These results suggest that the PDP technique may have clinical utility for modifying synthetic HAp for use as superior bone graft substitutes.     

Nano-sized ceramic particles of hydroxyapatite calcined with an anti-sintering agent

Nano-sized crystals of calcined hydroxyapatite (HAp) having spherical morphologies were fabricated by calcination at 800 degrees C for 1 h with an anti-sintering agent surrounding the original HAp particles and the agent was subsequently removed by washing after calcination. The original HAp particles were prepared by a modified emulsion system, and surrounded with poly(acrylic acid, calcium salt) (PAA-Ca) by utilizing a precipitation reaction between calcium hydroxide and poly(acrylic acid) adsorbed on the HAp particle surfaces in an aqueous medium. In the case of calcination without PAA-Ca, micron-sized particles consisting of sintered polycrystals were mainly observed by scanning electron microscopy, indicating the calcination-induced sintering among the crystals. On the other hand, most of the crystals calcined with the anti-sintering agent were observed as isolated particles, and the mean size of the HAp crystals was around 80 nm. This result indicates that PAA-Ca and its thermally decomposed product, CaO, surrounding the HAp crystals could protect them against calcination-induced sintering during calcination at 800 degrees C. The HAp crystals calcined with PAA-Ca showed high crystallinity, and no other calcium phosphate phases could be detected.     

New application of poly(butylene succinate) (PBS) based ionomer as biopolymer: a role of ion group for hydroxyapatite (HAp) crystal formation

Sodium sulfonate ionic group bearing PBS ionomer (PBSi) and hydroxyapatite (HAp) composites were prepared by soaking in the simulated body fluid (SBF) solution, which is the biomimetic method, as well as the effects of the ionic group on the HAp crystal formation and growth were investigated. The introduced sodium sulfonate ionic groups operated as the functional groups with negative charge densities, which can bind plentiful Ca²⁺ ions efficiently, consequently serving as active sites allowing HAp crystals to grow on the surfaces of the PBSi matrix. By SEM analysis, it was observed that HAp became growing as the shape with the porous holes. These holes are thought to be very suitable for the ingrowths of the surrounding tissue and the assistance to the bone formation. Based on this finding, it can be clearly concluded that the ionic groups in the PBSi may be decisive factors in growing HAp, and it is anticipated that this novel materials can contribute to excellent biopolymer.     

Influence of fluorine substitution on the morphology and structure of hydroxyapatite nanocrystals prepared by hydrothermal method

Hydroxyapatite (HAp) nanocrystals with different levels of fluorine substitution (P/F = 0, 6, 4 and 2) on the OH sites were produced via hydrothermal method. The fluorine substitution was found to alter the morphology of crystals appreciably. The aspect ratio and the crystallinity of HAp crystals increased with increasing fluorine substitution. The presence of broad ring and hallow ring patterns in electron diffraction suggests the low-crystalline nature of HAp crystals. With increasing fluorine substitution, the diffraction patterns exhibited discrete rings and numerous diffraction spots, implying the increased crystallinity. Raman spectra from the HAp nanoparticles also support the less-crystalline nature of the pristine HAp and the enhanced crystallization by fluorine substitution. In HAp crystals processed with no fluorine substitution, surface energy and planar Ca²⁺ density are less sensitive to the crystallographic orientation because of its low-crystalline nature, favoring equi-axed or slightly elongated particles. The addition of fluorine apparently increased the crystallinity, enhancing the orientation dependent growth and accordingly the aspect ratio. Osteoblast proliferation was observed to be enhanced by fluorine substitution in HAp. In vitro biological data support that the excellent osteoblastic cell viability and functional activity of the fluoridated apatite.     

Nano-scaled hydroxyapatite/polymer composite I. Coating of sintered hydroxyapatite particles on poly(γ-methacryloxypropyl trimethoxysilane)-grafted silk fibroin fibers through chemical bonding

The inorganic-organic composite consisting of nano-scaled hydroxyapatite (HAp) and silk fibroin (SF) fibers was prepared through covalent linkage to develop a novel biomaterial for a soft-tissue-compatible material. The preparation of the composite was conducted through the three-step procedure consisting of chemical modification using 2-methacryloxyethyl isocyanate (MOI) monomer to introduce vinyl groups on SF, poly(gamma-methacryloxypropyl trimethoxysilane) (MPTS) graft-polymerization on SF, and coupling process between the surface of polyMPTS-grafted SF and HAp nano-particles. The amount of the graft-polymerization of polyMPTS through vinyl groups was well controlled by the reaction time. The nano-crystals were subsequently coated on the grafted fibers by heating at 120 degrees C for 2 h in a vacuum. The crystalline structure of the SF substrate did not change in the procedure. In the SEM observation of the composite surface, it was found that the bonded nano-crystals were separated and partially aggregated with several crystals attached on the SF fiber surface. The HAp particles adhered more strongly on the SF surface with separation or aggregation of several crystals than on the surface of the original SF after ultrasonic treatment.     

Unidirectionally oriented hydroxyapatite/collagen composite fabricated by using a high magnetic field

A unidirectionally oriented hydroxyapatite (HAp)/collagen composite was prepared by using a high magnetic field. In the present study, a calcium-containing collagen solution was mixed with a phosphate-containing solution and kept at a temperature of 37 °C. During this process, the heat-induced self-assembling of collagen molecules and precipitation of HAp crystals took place simultaneously. Both collagen fibrils and HAp crystals have magnetic susceptibility anisotropy, which makes it possible for them to be oriented by using a high magnetic field. In order to prepare a unidirectionally oriented structure, samples were rotated when the high magnetic field was imposed perpendicularly to the rotation axis. The results indicated that collagen fibrils have been unidirectionally oriented with weakly crystallized HAp nanocrystals closely attached along the surfaces of the collagen fibrils. A biocomposite with similar structural and composition to natural bones has been prepared.     

Controlling Enamel Remineralization by Amyloid-Like Amelogenin Mimics

In situ regeneration of the enamel-like structure of hydroxyapatite (HAp) crystals under oral conditions is significant for dental caries treatment. However, it is still a challenge for dentists to duplicate the elegant and well-aligned apatite structure bonding to the surface of demineralized enamel. A biocompatible amelogenin-inspired matrix, a phase-transited lysozyme (PTL) film mimicking an N-terminal amelogenin with central domain (N-Ame) combined with synthetic peptide (C-AMG) based on the functional domains of C-terminal telopeptide (C-Ame) is shown here, which is formed by amyloid-like lysozyme aggregation at the enamel interface through a rapid one-step aqueous coating process. In the PTL/C-AMG matrix, C-AMG facilitated the oriented arrangement of amorphous calcium phosphate (ACP) nanoparticles and their transformation to ordered enamel-like HAp crystals, while PTL served as a strong interfacial anchor to immobilize the C-AMG peptide and PTL/C-AMG matrix on versatile substrate surfaces. PTL/C-AMG film-coated enamel induced both of the in vivo and in vitro synthesis of HAp crystals, facilitated epitaxial growth of HAp crystals and recovered the highly oriented structure and mechanical properties to levels nearly identical to those of natural enamel. This work underlines the importance of amyloid-like protein aggregates in the biomineralization of enamel, providing a promising strategy for treating dental caries.     

Hydrothermal conversion of calcite crystals to hydroxyapatite

Hydroxyapatite (HAp) has been successfully synthesized by hydrothermal treatment of calcite crystals in H₃PO₄ suspensions at 120 or 180 °C. Synthesis of HAp crystals under different H₃PO₄ concentrations and time conditions were studied. The results showed that HAp can be produced by the hydrothermal conversion of calcite crystals in the presence of a neutral suspension consisted of Ca 50 mM and PO₄ 60 mM, at 120 and 180 °C for 6 h. Unreacted CaCO₃ remained when PO₄ concentration was less than this at lower temperature. The major formation mechanism of HAp seems to be dissolution/precipitation.     

Effect of supersaturation on the morphology of hydroxyapatite crystals deposited by electrochemical deposition on titanium

Cathodic deposition of calcium phosphate coating on titanium using electrochemical method was investigated in our study and well-defined hexagonal rod-like hydroxyapatite (HAp) crystals were synthesized. Scanning electron microscopic photographs of HAp coatings under different deposition durations revealed the morphology change of the HAp crystals in the coating film with the experimental process: from cone-like structure to hexagonal prism with sharp-angled tip, and finally turns to rod-like one with regular hexagonal cross section. It was suggested that both morphology and longitudinal length of the HAp crystals in the coating could be regulated by an accurate control of the degree of supersaturation of the aqueous system.     

A study of the process and kinetics of electrochemical deposition and the hydrothermal synthesis of hydroxyapatite coatings

Hydroxyapatite (HAp) coatings were prepared using electrochemical deposition and post-hydrothermal synthesis. The composition and morphology of coatings at each processing step was studied through the application of scanning electron microscopy (SEM), X-ray diffraction (XRD) and infra-red spectroscopy (IR). The mechanism and kinetics of hydrothermal synthesis were considered in particular, and the influnce of the temperature and time on the HAp formation rate was also investigated. The results show that the electrochemical deposition coatings are composed of CaHPO₄2H₂O crystals which are converted into needle-like HAp crystals after post-hydrothermal treatment. The HAp content of the coatings increases with the treatment temperature and time. The synthesis rate also increases with the pH value of the water. The formation of HAp coatings is considered to be a combination of several reactions. An Arrhenius relationship was found between the HAp formation rate and the temperature, and an apparent activation energy of 94.4 KJ/mol was obtained by calculation. © 2000 Kluwer Academic Publishers     

Biomimetic synthesis of enamel-like hydroxyapatite on self-assembled monolayers

Hydroxyapatite (HAp) crystals mimicking tooth enamel in chemical composition and morphology were formed on sulfonic-terminated self-assembled monolayer (SAM) in 1.5SBF with F⁻ at 50 °C for 7 days. F⁻ ions showed a marked effect on the composition and morphology of deposited HAp crystals. In the absence of F⁻ ions, HAp containing CO₃²⁻ were formed on SAM, and worm-like crystals of 200–300 nm in length aggregated to form a spherical morphology. When F⁻ was added, HAp crystals containing both CO₃²⁻ and F⁻ were formed on SAM. Needle-shaped crystals of high aspect ratio and 1–2 μm in length grew elongated along the c-axial direction. In addition, these needle-shaped crystals grew in bundles, mimicking HAp crystals in tooth enamel. After the process of ripening, the needles in bundle grew to large size of up to 10 μm in length, and still kept no crystal–crystal fusion like enamel HAp crystals. The formation of enamel-like HAp can be attributed to the substitute of F⁻ for OH⁻ by disturbing the normal progress of HAp formation on SAM. The results suggest potential applications in preparing a novel dental material by a simple method.     

Development of the chitosan tube prepared from crab tendon for nerve regeneration

Crystalline chitosan was successfully prepared from crab tendons with aligned chitin molecules. Chitosan obtained preserved the tube structure with a suitable size for nerve conduits (t-chitosan tube). The chitosan tubes were compounded with hydroxy apatite (HAp) using an alternate soaking method (t-chitosan/HAp tube) to enhance the strength of the tube walls. HAp crystals formed in the walls of the chitosan tubes, and their c-axis aligned well in parallel with the chitosan molecules. The growth of the HAp crystals was found to occur at the nucleation sites, most probably by forming complexes with amino groups on chitosan mediated by Ca ions. Furthermore, these tubes were treated at 120 °C to prevent from swelling. These treatments preserved well the hollow nature of the tubes. And the mechanical test showed that the force was significantly higher in the t-chitosan/HAp tube when compared to the circular or triangular t-chitosan tube at each strain.Bridge grafting (15 mm) into the sciatic nerve of SD rats was carried out using the t-chitosan tubes having either a circular or triangular cross-section as well as t-chitosan/HAp tubes (N=18 in each group). Specimens were taken after two, four, six and eight weeks for histology (N=3 in each group). And nerve regeneration was evaluated histologically after 12 weeks (N=6 in each group). The t-chitosan/HAp tubes, demonstrated preferable biodegradation and biocompatibility. In addition to the mechanical properties of the tubes, the results of histological findings suggest that a triangular shape of the tube's cross-section and HAp coating may benefit nerve regeneration.     

Organic-inorganic interaction and the growth mechanism of hydroxyapatite crystals in gelatin matrices between 37 and 80 °C

The crystal development of hydroxyapatite[HAp] phase in gelatin[GEL] matrices was investigated in the temperature range 37 to 80 °C by using X-ray diffraction, scanning electron microscopy(SEM), thermoanalytical measurement(DT/TGA), Fourier-Transformed Infra-Red(FT-IR) spectroscopy, and transmission electron microscopy(TEM) with electron diffraction(ED). It was found that during the coprecipitation of apatite phase in GEL matrices and the next aging process the crystallites were formed and developed through the two reaction mechanisms of organic-inorganic interaction between apatite phase and GEL molecules, and thermodynamic reaction for the crystal growing. The analytical evidences showed that there was a definite competition between these two mechanisms with the reaction temperature. Below 50 °C the crystal development of HAp was greatly suppressed by the existence of the GEL molecules, indicating the heterogeneous nucleation by the supposed number of carboxyl groups in GEL. Above 50 °C the effective organic components as a template for the heterogeneous nucleation of apatite crystallites were greatly degraded and so more amount of inorganic ions could be favorably accredited on the preexisting crystallites in virtue of the limited nucleation chance, finally resulting in the crystal growth. At higher temperature pretty big HAp crystals were developed with the depletion of the organics to be bound with crystallites in the slurry solution. Presumably it is believed that the poisoning of the functional groups in GEL molecules was vigorously occurred in the phosphoric acid environment above ∼ 50 °C.     

A novel method for the fabrication of homogeneous hydroxyapatite/collagen nanocomposite and nanocomposite scaffold with hierarchical porosity

Homogeneous nanocomposites composed of hydroxyapatite (HAp) and collagen were synthesized using a novel in situ precipitation method through dual template-driven. The morphological and componential properties of nanocomposites were investigated. The HAp particulates, in sizes of about 50–100 nm, were distributed homogeneously in the organic collagen hydrogel. Highly magnified TEM observation showed that HAp inorganic particles were composed of fine sub-particles (2–5 nm) without regular crystallographic orientation. Based on these homogeneous nanocomposites, a novel HAp/collagen nanocomposite scaffold with hierarchical porosity was prepared by multilevel freeze-drying technique. Compared to other conventional scaffolds for tissue engineering, this novel in situ method endows synthesized composite scaffolds with unique morphology—ultrafine HAp particles dispersed homogenously in collagen at nano level and the foam scaffold with hierarchical pore structures. The mechanical performance increased obviously compared with neat collagen. These results provided an efficient approach toward new biomimetic tissue scaffold for the biomedical applications with enhanced intensity/bioactivity and controlled resorption rates. This novel method, we expect, will lead to a wide application in many other hydrogel systems and may be useful for fabrication of various homogeneous inorganic/organic nanocomposites.     

Transmission electron microscopic observation of a metastable phase on the thermal decomposition process of Ca-deficient hydroxyapatite

Calcium-deficient hydroxyapatite (Ca-def HAp) decomposes to stoichiometric hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) at high temperature. In a previous study, we reported that a metastable phase with a high Ca/P molar ratio appeared in the temperature range from 700 to 800°C. In the present study, the formation process of a metastable phase and the crystallographic relationship between the Ca-rich metastable phase and HAp matrix were investigated by high-resolution transmission electron microscopy (HRTEM). Ca-def HAp was annealed at 600–850°C for 2 or 6 h in air. TEM observations were performed before and after annealing Ca-def HAp. Based on analysis of image of Ca-def HAp before annealing, several HAp crystals with different aspect ratios agglomerated. The metastable phases grew thicker by long-term annealing. HRTEM image suggested that the Ca-rich metastable phase was formed by migration to the interface and continuous accumulation of calcium ions from HAp crystals with a small aspect ratio. From HRTEM images and results of the analysis of selected area electron diffraction patterns along the [010], [110] and [001] zone axes, lattice constants of the metastable phases were determined to be a = 2.86 nm, b = 0.94 nm, and c = 0.69 nm with orthorhombic crystals system.     

Preparation of hydroxyapatite/poly(methyl methacrylate) and calcium silicate/poly(methyl methacrylate) interpenetrating hybrid composites

Hydroxyapatite/poly(methyl methacrylate) (HAp/PMMA) and calcium silicate/poly(methyl methacrylate) (CS/PMMA) composites were prepared by interpenetrating bulk polymerization of methyl methacrylate (MMA) monomer in porous structures of HAp and CS. The porous HAp and CS templates were prepared by mixing their calcined powders with poly(vinyl alcohol) (PVA) solution, shaping by uniaxial pressing and then firing at 1,100 °C for HAp and 900 °C for CS. The templates were soaked in the solution mixture of MMA monomer and 0.1 mol% of benzoyl peroxide (BPO) for 24 h. The pre-composites were then bulk polymerized at 85 °C for 24 h under nitrogen atmosphere. The microstructures of the composites showed the interpenetrating of PMMA into the porous HAp and CS structures. Thermogravimetric analysis indicated that the PMMA content in the HAp/PMMA and CS/PMMA composites were 13 and 26 wt%, respectively. Weight average molecular weights ( ) of PMMA were about 491,000 for HAp/PMMA composites and about 348,000 for CS/PMMA composites. Compressive strengths of these composites were about 90–131 MPa in which they were significantly higher than their starting porous templates.     

Effect of urea on formation of hydroxyapatite through double-step hydrothermal processing

The effect of urea on the formation of hydroxyapatite (HAp) was studied by employing the double-step hydrothermal processing of a powder mixture of beta-tricalcium phosphate (β-TCP) and dicalcium phosphate dihydrate (DCPD). Co-existence of urea was found to sustain morphology of HAp crystals in the compacts under an initial concentration of 2 mol dm^-3 and less. Homogenous morphology of needle-like crystals was observed on the compacts carbonated owing to decomposition of urea. Carbonate ions (CO₃^2-) was found to be substituted in both the phosphate and hydroxide sites of HAp lattice. The synthesized HAp was calcium deficient, as it had a Ca/P atomic ratio of 1.62 and the phase was identified as calcium deficient hydroxyapatite (CDHA). The release of CO₃^2- ions from urea during the hydrothermal treatment determined the morphology of the CDHA in the compacts. The usage of urea in the morphological control of carbonate-substituted HAp (CHAp) employing the double-step hydrothermal method is established.     

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.     

Preparation of gold/hydroxyapatite hybrids using natural fish scale template and their effective albumin interactions

The gold (Au) nanoparticles (NPs) with the diameter of 15–40?nm were successfully synthesized in the hierarchical hydroxyapatite (HAp) nanostructures of natural fish scale templates, which were carried out by the Au³⁺ ion chemisorption, reduction and calcination processes to form the AuNPs/HAp hybrids. The AuNPs size as well as the surface plasmon resonance (SPR) absorption maximum was preserved with the hybridization process. Moreover, the AuNPs/HAp hybrid nanostructures exhibited preferential protein adsorption behavior at the biological bovine serum albumin (Ab) concentration regions that correspond to be 1.5?μM in the cell culture medium and 15.1?μM in human blood, and the Ab adsorption equilibrium constant of AuNPs/HAp hybrid was higher than that of the HAp alone. The SPR absorption maxima of the Ab-adsorbed AuNPs/HAp fish scales were red-shifted as compared with those of the AuNPs/HAp fish scales. Therefore, we synthesized the AuNPs using the fish scale template to exhibit the preferential protein adsorption, which will be a great significance to research the AuNPs/HAp hybrid functions.     

Preparation of anatase nanocrystals deposited on hydroxyapatite by hydrothermal treatment

TiO₂-deposited hydroxyapatite (HAp) crystals have been successfully synthesized by hydrothermal treatment of nearly neutral suspension of HAp powders in 10 vol.% of TAS-FINE™ (Titanium amine complex) solutions at 120 or 180 °C. Resultant products were characterized by XRD, SEM–EDX, Vis-Raman and TEM. SEM and TEM observation showed that small rodlike TiO₂, identified as anatase crystals of 100–150 nm in length, were deposited on HAp plates/crystals. The anatase crystals seem to be formed by heterogeneous nucleation on the surfaces of HAp crystals. A higher temperature and a longer reaction period promoted the cystallization of anatase TiO₂.