Studies of early growth mechanisms of hydroxyapatite on single crystalline rutile: a model system for bioactive surfaces

Previous studies have shown that crystalline titanium oxide is in vitro bioactive and that there are differences in the HA formation mechanism depending on the crystalline direction of the titanium oxide surface. In the present study, the early adsorption of calcium and phosphate ions on three different surface directions of the single-crystal rutile TiO₂ substrate has been investigated. A crucial step in the nucleation of HA is believed to be the adsorption of Ca²⁺ and PO₄ ³⁻ from phosphate buffer solutions. The (001), (100) and (110) single crystalline rutile surfaces were soaked in phosphate buffer saline solution for 10 min, 1 h and 24 h at 37°C. The surfaces were then analyzed using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS). The results show that the adsorption of Ca²⁺ and PO₄ ³⁻ is faster on the (001) and (100) surfaces than on the (110) surface. This study also shows that TOF-SIMS can be used as a tool to better understand the adsorption of calcium and phosphate ions and the growth mechanism of HA. This knowledge could be used to tailor new bioactive surfaces for better biological reaction.     

Synthesized silicon-substituted hydroxyapatite coating on titanium substrate by electrochemical deposition

Silicon-substituted hydroxyapaptite (Si-HA) coatings were prepared on titanium substrates by electrolytic deposition technique in electrolytes containing Ca²⁺, PO₄ ³⁻ and SiO₃ ²⁻ ions with various SiO₃ ²⁻/(PO₄ ³⁻ + SiO₃ ²⁻) molar ratios(η_si). The deposition was all conducted at a constant voltage of 3.0 V, with titanium substrate as cathode and platinum as anode, for 1 h at 85°C. The coatings thus prepared were characterized with inductively coupled plasma (ICP), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), field-emission-type scanning electron microscope (FSEM). The results show that the silicon amount in the coatings increases linearly to about 0.48 wt% at first with increasing η_si between 0 and 0.03, then increases slowly to about 0.55 wt% between 0.03 and 0.10 and finally maintains almost at a level around 0.55 wt% between 0.10 and 0.30. The tree-like Si-HA crystals are observed in the coatings prepared in the electrolyte of η_si = 0.20. And the presence of silicon in electrolytes decreases the thickness of the coatings, with effect being more significant as η_si increased. Additionally, the substitution of Si causes some OH⁻ loss and changes the lattice parameters of hydroxyapatite (HA).     

Hydroxyapatite-collagen composites. Part I: can the decrease of the interactions between the two components be a physicochemical component of osteoporosis in aged bone?

The interactions of Type I acid soluble collagen (Col) with both carbonate-free hydroxyapatite (HA₁₁₀₀) and carbonate-rich one (CHA) were investigated. The aim was to ascertain whether the increase of bone CO₃ ²⁻ with ageing could relate to the disease known as osteoporosis. HA₁₁₀₀-Col and CHA-Col composites with various ratios were prepared and examined. Scanning electron microscopy and differential scanning calorimetry showed a stronger adhesion of the Col matrix to the granules of HA₁₁₀₀ than to those of CHA. FT-IR spectroscopy showed that with HA₁₁₀₀ both multiple hydrogen bonds of Col peptide –NH groups with HA PO₄ ³⁻, and electrochemical interactions between Col peptide –C=O groups and HA Ca²⁺ were present. In the presence of CO₃ ²⁻, the interactions between –NH and phosphate were diminished, and Ca²⁺ interacted more strongly with CO₃ ²⁻ than with peptide –C=O, so causing a separation between the two components of the bone extra-cellular matrix. The results obtained strengthen the hypothesis that the substitution of PO₄ ³⁻ ions by CO₃ ²⁻ ions in the HA lattice might be a significant component of osteoporosis, although further investigation is needed.     

In vitro behavior of fluoridated hydroxyapatite coatings in organic-containing simulated body fluid

A dense and pure hydroxyapatite [HA, Ca₁₀(PO₄)₆(OH)₂] coating and a fluoridated HA [Ca₁₀(PO₄)₆(OH)_0.67F_1.33] are deposited on Ti6Al4V substrates by sol-gel dip coating method. Glucose and bovine serum albumin have been added in standard simulated body fluid (SBF) to form organic-containing SBF in simulation of the physiological blood plasma. The HA and the fluoridated HA coatings are immersed in the standard and modified SBF for time periods of 2, 4, 7, 14 and 28 days at 37 ± 0.1°C. After soaking, the coating surface is examined for nucleation and growth of apatite using SEM morphological observation. The post-soaking SBF solutions are analyzed via Inductively Coupled Plasma spectroscopy for calcium ion concentration. The results show that at concentration of 40 g/L, bovine serum albumin has significant retardation effect on apatite precipitation from SBF onto pure or fluoridated HA coatings; Fluorine-incorporation in HA has positive bio-activation effect in both standard SBF and organic-containing SBF. However, glucose addition in SBF does not generate significant influence on the bioactivity of HA and fluoridated HA.     

Hydrothermal precipitation of hydroxyapatite on anodic titanium oxide films containing Ca and P

Highly-crystallized hydroxyapatite (HA) can be precipitated during heat treatment in high-pressure steam at 300 °C on an anodic titanium oxide film containing Ca and P (AOFCP), which has been electrochemically formed on a titanium substrate prior to the hydrothermal treatment. Factors affecting the precipitation, such as a percentage of distilled water in the autoclave and additives in the AOFCP, were evaluated by scanning electron microscopy. Ca²⁺ and PO^3– ₄ ions were leached from the AOFCP into a water layer covering the film surface, and nucleate HA heterogeneously on the porous TiO₂ matrix of the AOFCP which was made by the ion leaching. The morphology of the precipitated crystals was significantly affected by the water volume ratio because the concentrations of the Ca²⁺ and PO₄ ^3– ions varied depending on the thickness of the water layer. The amount of the precipitation decreased on the AOFCP which was formed in the solution containing a small amount of Mg²⁺ ions or formed on Ti-6Al-4V alloy instead of titanium.     

Silicon-substituted hydroxyapatite composite coating by using vacuum-plasma spraying and its interaction with human serum albumin

The incorporation of silicon can improve the bioactivity of hydroxyapatite (HA). Silicon-substituted HA (Ca₁₀(PO₄)_6−x (SiO₄) _x (OH)_2−x , Si-HA) composite coatings on a bioactive titanium substrate were prepared by using a vacuum-plasma spraying method. The surface structure was characterized by using XRD, SEM, XRF, EDS and FTIR. The bond strength of the coating was investigated and XRD patterns showed that Ti/Si-HA coatings were similar to patterns seen for HA. The only different XRD pattern was a slight trend toward a smaller angle direction with an increase in the molar ratio of silicon. FTIR spectra showed that the most notable effect of silicon substitution was that –OH group decreased as the silicon content increased. XRD and EDS elemental analysis indicated that the content of silicon in the coating was consistent with the silicon-substituted hydroxyapatite used in spraying. A bioactive TiO₂ coating was formed on an etched surface of Ti, and the etching might improve the bond strength of the coatings. The interaction of the Ti/Si-HA coating with human serum albumin (HSA) was much greater than that of the Ti/HA coating. This might suggest that the incorporation of silicon in HA can lead to significant improvements in the bioactive performance of HA.     

Synthesis and cytocompatibility of manganese (II) and iron (III) substituted hydroxyapatite nanoparticles

Manganese (II) and iron (III) substituted hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂) nanoparticles were synthesized using wet chemical method. All samples were single-phase, non-stoichiometric and B-type carbonated hydroxyapatite. Compared with pure HA, Mn²⁺ substituted (MnHA) and Fe³⁺ doped HA (FeHA) did not demonstrate significant structure deviation. Since ion exchange mechanism was applied for the synthesis process, the morphology and particle size were not significantly affected: all samples were elongated spheroids of around 70 nm. The presence of Fe and Mn was confirmed by energy dispersive X-ray spectroscopy (EDX) while the concentrations were quantified by inductively coupled plasma (ICP). Fe³⁺ ions were more active than Mn²⁺ ions in replacing Ca²⁺ ions in HA lattice structure. The magnetic property of HA was modified by substitution with Fe. The Fe5 (Fe_added/Ca_added = 5% by molar ratio) was paramagnetic while pure HA was diamagnetic. Results of extraction assay from cells cultured in extracted medium for 72 h suggested that both MnHA and FeHA were non-cytotoxic to osteoblast cells. Meanwhile, the presence of Fe³⁺ ions in HA demonstrated significant positive effect on osteoblast cells, where the cell number on Fe5 pellets was twice that of pure HA and MnHA samples.     

Preparation of highly porous hydroxyapatite from cuttlefish bone

Hydroxyapatite structures for tissue engineering applications have been produced by hydrothermal (HT) treatment of aragonite in the form of cuttlefish bone at 200°C. Aragonite (CaCO₃) monoliths were completely transformed into hydroxyapatite after 48 h of HT treatment. The substitution of CO₃ ²⁻ groups predominantly into the PO₄ ³⁻ sites of the Ca₁₀(PO₄)₆(OH)₂ structure was suggested by FT-IR spectroscopy and Rietveld structure refinement. The intensity of the ν₃PO₄ ³⁻ bands increase, while the intensity of the ν₂CO₃ ²⁻ bands decrease with the duration of HT treatment resulting in the formation of carbonate incorporating hydroxyapatite. The SEM micrographs have shown that the interconnected hollow structure with pillars connecting parallel lamellae in cuttlefish bone is maintained after conversion. Specific surface area (S _BET) and total pore volume increased and mean pore size decreased by HT treatment.     

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 MPam^1/2, respectively.     

Hydrothermal synthesis of calcium hydroxyapatite nanorods in the presence of PVP

Calcium hydroxyphosphate (Ca₁₀(PO₄)₆(OH)₂, HAP) nanorods have been successfully synthesized by a simple and mild hydrothermal treatment in the presence of polyvinylpyrrolidone (PVP). A complex of calcium nitrate (Ca(NO₃)₂) and Na₂HPO₄ was used to supply the calcium and phosphate ions during the reactions. The synthesis of pure HAP nanorods was under near neutral condition. The morphology and structure of the samples were characterized by transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) spectroscopy analysis. The nanorods were uniform with diameter of 20–25 nm and length ranging from several hundreds of nanometers to several micrometers. The influence of different experiment conditions, i.e., the PVP concentration, molar ratio of Ca²⁺ to HPO₄ ²⁻, reaction time, and temperature, on the morphology of the nanorods was investigated. The formation mechanism of rod-like HAP and effects of PVP on the crystal nucleation and growth have also been discussed.     

Deposition of bone-like hydroxyapatite on the surface of silk cloth with the aid of immobilized urease

Continuous layers of hydroxyapatite were deposited on silk cloth from aqueous solutions by using urease as the precipitant supplier. Silk cloth was surface-modified with urease and was immersed in an aqueous solution containing Ca²⁺, PO₄³⁻, and urea. As urea was hydrolyzed to form ammonia with the aid of the immobilized urease, hydroxyapatite precipitated predominantly on the surface of the silk cloth. It took only a few hours to form continuous layers of hydroxyapatite on the silk cloth. The resultant hydroxyapatite was found to be bone-like apatite because it had low crystallinity, contained carbonate ion in the lattice, and had a calcium-deficient composition.     

Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions

Bioactive glasses with controllable conversion rates to hydroxyapatite (HA) may provide a novel class of scaffold materials for bone tissue engineering. The objective of the present work was to comprehensively characterize the conversion of a silicate bioactive glass (45S5), a borate glass, and two intermediate borosilicate glass compositions to HA in a dilute phosphate solution at 37°C. The borate glass and the borosilicate glasses were derived from the 45S5 glass by fully or partially replacing the SiO₂ with B₂O₃. Higher B₂O₃ content produced a more rapid conversion of the glass to HA and a lower pH value of the phosphate solution. Whereas the borate glass was fully converted to HA in less than 4 days, the silicate (45S5) and borosilicate compositions were only partially converted even after 70 days, and contained residual SiO₂ in a Na-depleted core. The concentration of Na⁺ in the phosphate solution increased with reaction time whereas the PO₄^3– concentration decreased, both reaching final limiting values at a rate that increased with the B₂O₃ content of the glass. However, the Ca²⁺ concentration in the solution remained low, below the detection limit of atomic absorption, throughout the reaction. Immersion of the glasses in a mixed solution of K₂HPO₄ and K₂CO₃ produced a carbonate-substituted HA but the presence of the K₂CO₃ had little effect on the kinetics of conversion to HA. The kinetics and mechanisms of the conversion process of the four glasses to HA are compared and used to develop a model for the process.     

Monitoring of hydroxyapatite conversion by luminescence intensity of Eu3+ ions during mineralization of Eu3+-doped β-Ca2SiO4

β-Dicalcium silicate (β-Ca₂SiO₄) doped with Eu³⁺ was synthesized by sol–gel method. The luminescence intensity of the mineralization products formed during the hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HA) conversion of Eu³⁺-doped β-Ca₂SiO₄, in 0.25 M K₂HPO₄ solution, were detected using luminescence spectroscopy. The results indicated that the luminescence intensity of Eu³⁺ ion gradually depressed with prolonged mineralization time, and it could hardly be detected with the complete transformation from β-Ca₂SiO₄:Eu³⁺ to hydroxyapatite. The change of Eu³⁺ ionic concentrations in the mineralization products and the final solutions after conversion reaction, were further examined using energy-dispersive X-ray and inductively-coupled plasma mass spectrometry, respectively. This suggested that the process of mineralization can be monitored with the luminescence intensity of Eu³⁺ ions in the mineralization products. The current study will open up a new and simple in vivo avenue for in situ monitoring hydroxyapatite conversion with a fiber luminescence spectrometer.     

Magnesium- and strontium-co-substituted hydroxyapatite: the effects of doped-ions on the structure and chemico-physical properties

The present study is aimed at investigating the contribution of two biologically important cations, Mg²⁺ and Sr²⁺, when substituted into the structure of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂,HA). The substituted samples were synthesized by an aqueous precipitation method that involved the addition of Mg²⁺- and Sr²⁺-containing precursors to partially replace Ca²⁺ ions in the apatite structure. Eight substituted HA samples with different concentrations of single (only Mg²⁺) or combined (Mg²⁺ and Sr²⁺) substitution of cations have been investigated and the results compared with those of pure HA. The obtained materials were characterized by X-ray powder diffraction, specific surface area and porosity measurements (N₂ adsorption at 77?K), FT-IR and Raman spectroscopies and scanning electron microscopy. The results indicate that the co-substitution gives rise to the formation of HA and ??-TCP structure types, with a variation of their cell parameters and of the crystallinity degree of HA with varying levels of substitution. An evaluation of the amount of substituents allows us to design and prepare BCP composite materials with a desired HA/??-TCP ratio.     

Synthesis of nanocrystalline fluorinated hydroxyapatite by microwave processing and its in vitro dissolution study

Synthetic hydroxyapatite, (Ca₁₀(PO₄)₆(OH)₂, HA), is an important material used for orthopedic and dental implant applications. The biological hydroxyapatite in the human bone and tooth is of nanosize and differs in composition from the stoichiometric HA by the presence of other ions such as carbonate, magnesium, fluoride, etc. Osseointegration is enhanced by using nanocrystalline HA. This stimulates the interest in synthesizing nanocrystalline HA by different routes and among the methods, microwave processing seems to form the fine grain size and uniform characteristic nanocrystalline materials. Fluorinated hydroxyapatite, (FHA, Ca₁₀(PO₄)₆(OH)_2−x F _x ), possesses higher corrosion resistance in biofluids than pure HA and reduces the risk of dental caries. The present work deals with the synthesis of nanocrystalline FHAs by microwave processing. The crystal size and morphology of the nanopowers were examined by X-ray powder diffraction (XRD) and transmission electron microscopy (TEM) methods. The functional groups present in FHA powders were ascertained by Fourier transform infrared spectroscopy (FT-IR) and laser Raman spectroscopy. Since the physiological stability is an important parameter while selecting the material for implantation, the in vitro dissolution studies of FHAs with different fluorine contents were carried out.     

Crystal structure and luminescent properties of europium-activated Ca10? x M x (PO4)6F2 (M = Pb, Mg) prepared via precipitation from aqueous solutions

Phase-pure polycrystalline fluorapatites with the general formula Ca_10−x M _x (PO₄)₆F₂:Eu³⁺(M = Pb, Mg) have been prepared by precipitation from aqueous solutions, and the effects of the Pb²⁺ and Mg²⁺ ions, differing markedly in ionic radius, on the structure, morphology, and luminescence spectra of the fluorapatites have been investigated. The Pb²⁺ and Mg²⁺ contents are shown to influence the Eu³⁺ distribution over inequivalent sites in the crystal structure of Ca_10−x M _x (PO₄)₆F₂. Original Russian Text ? N.V. Babayevskaya, Yu.N. Savvin, A.V. Tolmachev, 2007, published in Neorganicheskie Materialy, 2007, Vol. 43, No. 8, pp. 976–980.     

Characterization of sintered titanium/hydroxyapatite biocomposite using FTIR spectroscopy

Fourier transform infrared (FTIR) spectroscopy was employed to characterize the phase changes of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HA) in a titanium/HA biocomposite during sintering. The effects of sintering temperature and the presence of Ti on the decomposition of HA were examined. It was observed that pure HA was stable in argon atmosphere at temperatures up to 1,200°C, although the dehydroxylation of pure HA was promoted by the increase in sintering temperature. In the Ti/HA system, on the other hand, the presence of Ti accelerated dehydroxylation and the decomposition of HA was detected at a temperature as low as 800°C. Tetracalcium phosphate (Ca₄P₂O₉, TTCP) and calcium oxide (CaO) were the dominant products of the decomposition, but no tricalcium phosphate (Ca₃(PO₄)₂, TCP) was detected due to phosphorus diffusion and possible reactions during the thermal process. The main decomposed constituents of HA in Ti/HA system at high temperatures (≥1,200°C) would be CaO and amorphous phases.     

Hydrothermal preparation of tailored hydroxyapatite

Hydrothermal vapor treatment method was applied for preparation of ceramic biomaterials. Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂; HA) ceramics prepared by sintering with random crystal surface have already been used as bone-repairing materials which can directly bond to natural bones. If materials of HA could have the tailored specific crystal surface, they should have the advantage of adsorptive activity and osteoconductivity in comparison with the sintered HA. In the present study, porous HA sheets of about 50 μm to 1 mm in thickness and porous HA granules of about 50 μm to 1 mm in size with tailored crystal surface were prepared by the hydrothermal vapor exposure method at temperatures below 200°C. Porous sheets and porous granules of HA with controlled crystal surface should be suitable for scaffold of cultured bone, for bone graft material and for drug delivery system (DDS).     

Ribbon-like and rod-like hydroxyapatite crystals deposited on titanium surface with electrochemical method

Homogeneous coatings were attained by electrochemical method in electrolytes containing Ca²⁺ and PO₄³⁻ ions with Ca/P ratio being 1.67. SEM observation showed that the hydroxyapatite (HAp,Ca₁₀(PO₄)₆(OH)₂) crystals prepared with higher concentration electrolyte (4 × 10^− 2 M Ca²⁺) are ribbon-like with thickness of nanometer size, a morphology seldom reported previously. In an electrolyte of lower concentration (6 × 10^− 4 M Ca²⁺), the HAp crystals formed are rod-like with a hexagonal cross section and diameter of about 70–80 nm. XRD patterns and IR spectra confirmed that the coatings consist of HAp crystals. TEM micrographs and SAD indicated that the longitude direction for both ribbon-like and rod-like crystal is [002], and the flat surface of the ribbon is (110). HRTEM showed that the ribbon-like crystal is a mixture of HAp and octacalcium phosphate (OCP, Ca₈H₂(PO₄)₆.5H₂O).     

Microwave annealing for preparation of crystalline hydroxyapatite thin films

A sol was spun on single crystal silicon substrates at a spin-rate of 3000–5000 rpm followed by a low temperature cure to form a stable sol–gel/silicon structure. Good quality crystalline HA films of thickness ∼300–400 nm were obtained by annealing the sol–gel/Si structure in a conventional cavity applicator microwave system with a magnetron power of 1300 W, frequency of 2.45 GHz, and at a low processing temperature of 425 °C for annealing times ranging from 2–60 min. X-ray Diffraction and FTIR analysis confirmed that the crystalline quality of the thin films were comparable or better than those heat-treated under the same processing conditions (temperature and time) in a Rapid Thermal Annealing (RTA) system. The RBS data suggests a composition corresponding to stoichiometric hydroxyapatite Ca₁₀(PO₄)₆(OH)₂, the major inorganic component of bone. The results showed that the HA film thickness decreases with increasing sol spin-rate. The HA films showed good biocompatibility because little monocyte adhesion occurred and hence no inflammatory response was activated in vitro. The potential of microwave annealing for rapid and low temperature processing of good crystalline quality HA thin films derived from sol–gel is demonstrated.