TEM-EDX study of mechanism of bonelike apatite formation on bioactive titanium metal in simulated body fluid

Bioactive titanium metal, which forms a bonelike apatite layer on its surface in the body and bonds to the bone through the apatite layer, can be prepared by NaOH and heat treatments to form an amorphous sodium titanate layer on the metal. In the present study, the mechanism of apatite formation on the bioactive titanium metal has been investigated in vitro. The metal surface was examined using transmission electron microscopy and energy dispersive X-ray spectrometry as a function of the soaking time in a simulated body fluid (SBF) and complemented with atomic emission spectroscopy analysis of the fluid. It was found that, immediately after immersion in the SBF, the metal exchanged Na(+) ions from the surface sodium titanate with H(3)O(+) ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after they were formed, incorporated the calcium ions in the fluid to form an amorphous calcium titanate. After a long soaking time, the amorphous calcium titanate incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate with a low Ca/P atomic ratio of 1.40. The amorphous calcium phosphate thereafter converted into bonelike crystalline apatite with a Ca/P ratio of 1.65, which is equal to the value of bone mineral. The initial formation of the amorphous calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania, which are dissociated from the Ti-OH groups, with the positively charged calcium ions in the fluid. The amorphous calcium titanate is speculated to gain a positive charge and to interact with the negatively charged phosphate ions in the fluid to form the amorphous calcium phosphate, which eventually stabilizes into bonelike crystalline apatite.     

Process and kinetics of bonelike apatite formation on sintered hydroxyapatite in a simulated body fluid

The surfaces of two hydroxyapatites (HA), which have been sintered at different temperatures of 800 and 1200 degrees C, was investigated as a function of soaking time in simulated body fluid (SBF) using transmission electron microscopy (TEM) attached with energy-dispersive spectrometry (EDX) and laser electrophoresis spectroscopy. The TEM-EDX indicated that after soaking in SBF, both the HAs form bonelike apatite by undergoing the same surface structural change, i.e., formations of a Ca-rich amorphous or nano-crystalline calcium phosphate (ACP) and a Ca-poor ACP, which eventually crystallized into bonelike apatite. Zeta potential characterized by the electrophoresis indicated that during exposure to SBF, the HA surfaces reveal negative surface charge, thereby interacting with the positive calcium ions in the fluid to form the Ca-rich ACP, which gains positive surface charge. The Ca-rich ACP on the HAs then interacts with the negative phosphate ions in the fluid to form the Ca-poor ACP, which stabilizes by being crystallized into bonelike apatite with a low solubility in the SBF. The exposure times for formations of these phases of the Ca-rich ACP, the Ca-poor ACP as well as the apatite were, however, all late on HA sintered at 1200 degrees C, compared with the HA sintered at 800 degrees C. This phenomenon was attributed to a lower initial negative surface charge of the HA sintered at 800 degrees C than of that one sintered at 1200 degrees C, owing to poverty in surface hydroxyl and phosphate groups which are responsible for the surface negativity of the HA. These indicate that sintered temperature of HA might influence not in terms of the process but in terms of the rate of formation of biologically active bonelike apatite on its surface, through which the HA integrates with living bone.     

Surface potential change in bioactive titanium metal during the process of apatite formation in simulated body fluid

Bioactive titanium metal can be prepared by NaOH and heat treatments that present the metal with a graded bioactive surface layer of amorphous sodium titanate. This study used laser electrophoresis together with transmission electron microscopy (TEM) and energy-dispersive X-ray microanalysis (EDX) to relate the surface potential change of the bioactive titanium metal with its surface structural change in simulated body fluid (SBF). The surface potential of the metal was highly negative immediately after immersion in SBF. With increasing soaking time, the surface potential increased, revealing a maximum positive value, and then decreased to a constant negative value. TEM-EDX showed that immediately after immersion in SBF, the metal surface formed Ti-OH groups by exchanging Na(+) ions in the surface sodium titanate with H3O(+) ions in the fluid. Thereafter, with increasing soaking time the metal surface formed an amorphous calcium titanate, then an amorphous calcium phosphate, and, finally, apatite with bone-like composition and structure. These results indicate that the process of apatite formation on bioactive titanium metal is initiated by the formation of Ti-OH groups with negative charges that interact with calcium ions with positive charges to form calcium titanate. The calcium titanate gains a positive charge and later interacts with phosphate ions with negative charges, forming amorphous calcium phosphate. The amorphous calcium phosphate eventually transforms and stabilizes into bone-like crystalline apatite with a negative charge.     

Structural dependence of apatite formation on titania gels in a simulated body fluid

The apatite-forming ability of titania gels with different structures has been investigated in a simulated body fluid with ion concentrations nearly equal to those of human blood plasma. Titania gels with an amorphous structure or with an anatase or rutile structure were prepared by the sol-gel process with a subsequent heat treatment at various temperatures. The titania gels with an amorphous structure did not induce apatite formation on their surfaces in the simulated body fluid, whereas gels with an anatase or rutile structure induced apatite formation on their surfaces. The deposition of apatite was more pronounced on the anatase gels than on the rutile gels. This indicates that a specific structure of titania is effective in inducing apatite formation in a body environment. Such a specific structure was assumed in this study to be the crystalline planar arrangement in the anatase structure, which facilitates epitaxy of the apatite crystal.     

Mechanism of apatite formation on wollastonite coatings in simulated body fluids

The formation mechanism of apatite on the surface of wollastonite coating was examined. Plasma-sprayed wollastonite coatings were soaked in a lactic acid solution (pH=2.4) to result in the dissolution of calcium from the coating to form silanol (triple bond Si-OH) on the surface. Some calcium-drained samples were soaked in a trimethanol aminomethane solution (pH=10) for 24h to create a negatively charged surface with the functional group (triple bond Si-O(-)). These samples before and after treatment in a trimethanol aminomethane solution were immersed in simulated body fluids (SBF) to investigate the precipitation of apatite on the coating surface. The results indicate that the increase of calcium in the SBF solution is not the critical factor affecting the precipitation of apatite on the surface of the wollastonite coating and the apatite can only form on a negatively charged surface with the functional group (triple bond Si-O(-)). The mechanism of apatite formation on the wollastonite coating is proposed. After the wollastonite coatings are immersed into the SBF, calcium ions initially exchange with H(+) leading to the formation of silanol (triple bond Si-OH) on the surface of the layer and increase in the pH value at the coating-SBF interface. Consequently, a negatively charged surface with the functional group (triple bond Si-O(-)) forms on the surface. Due to the negatively charged surface, Ca(2+) ions in the SBF solution are attracted to the interface between the coating and solution, thereby increasing the ionic activity of the apatite at the interface to the extent that apatite precipitates on the coating surface.     

Fluoride-containing bioactive glasses: Effect of glass design and structure on degradation,pH and apatite formation in simulated body fluid

Bioactive glasses are able to bond to bone through formation of carbonated hydroxyapatite in body fluids, and fluoride-releasing bioactive glasses are of interest for both orthopaedic and, in particular, dental applications for caries inhibition. Melt-derived glasses in the system SiO₂–P₂O₅–CaO–Na₂O with increasing amounts of CaF₂ were prepared by keeping network connectivity and the ratio of all other components constant. pH change, ion release and apatite formation during immersion of glass powder in simulated body fluid at 37 °C over up to 2 weeks were investigated. Crystal phases formed in SBF were characterized using infrared spectroscopy, X-ray diffraction with Rietveld analysis and solid-state nuclear magnetic resonance spectroscopy (¹⁹F and ³¹P MAS–NMR). Results show that incorporation of fluoride resulted in a reduced pH rise in aqueous solutions compared to fluoride-free glasses and in formation of fluorapatite (FAp), which is more chemically stable than hydroxyapatite or carbonated hydroxyapatite and therefore is of interest for dental applications. However, for increasing fluoride content in the glass, fluorite (CaF₂) was formed at the expense of FAp. Apatite formation could be favoured by increasing the phosphate content in the glass, as the release of additional phosphate into the SBF would affect supersaturation in the solution and possibly favour formation of apatite.     

Surface functional group dependent apatite formation on bacterial cellulose microfibrils network in a simulated body fluid

The apatite forming ability of biopolymer bacterial cellulose (BC) has been investigated by soaking different BC specimens in a simulated body fluid (1.5 SBF) under physiological conditions, at 37 degrees C and pH 7.4, mimicking the natural process of apatite formation. From ATR-FTIR spectra and ICP-AES analysis, the crystalline phase nucleated on the BC microfibrils surface was calcium deficient carbonated apatite through initial formation of octacalcium phosphate (OCP) or OCP like calcium phosphate phase regardless of the substrates. Morphology of the deposits from SEM, FE-SEM, and TEM observations revealed the fine structure of thin film plates uniting together to form apatite globules of various size (from <1 mum to 3 mum) with respect to the substrates. Surface modification by TEMPO (2,2,6,6-tetramethylpyperidine-1-oxyl)-mediated oxidation, which can readily form active carboxyl functional groups upon selective oxidation of primary hydroxyl groups on the surface of BC microfibrils, enhanced the rate of apatite nucleation. Ion exchanged treatment with calcium chloride solution after TEMPO-mediated oxidation was found to be remarkably different from other BC substrates with the highest deposit weight and the smallest apatite globules size. The role of BC substrates to induce mineralization rate differs according to the nature of the BC substrates, which strongly influences the growth behavior of the apatite crystals.     

Bonelike apatite growth on hydroxyapatite-gelatin sponges from simulated body fluid.

In vitro bioactivity of gelatin sponges and hydroxyapatite-enriched gelatin sponges was tested through evaluation of the variations in their composition and morphology after soaking in simulated body fluid (1.5) for periods up to 21 days at 37 degrees C. The presence of hydroxyapatite inside the sponges promotes the deposition of bonelike apatite crystals. The deposits are laid down as spherical aggregates, with mean diameters increasing from about 1-2 microm, after 4 days of soaking in simulated body fluid solution, up to about 3.5 microm in the samples soaked for 21 days. Simultaneously, the relative amount of inorganic phase increases up to about 56% wt, leading to a composite material with a composition quite close to that of bone tissue. The inorganic phase is a poor crystalline carbonated apatite similar to trabecular bone apatite.     

Effect of wollastonite ceramics and bioactive glass on the formation of a bonelike apatite layer on a cobalt base alloy

A biomimetic method was used to promote a bioactive surface on a cobalt base alloy (ASTM F-75). The metallic substrates were alkali treated and some of the samples were subsequently heat treated. The treated samples were immersed in simulated body fluid (SBF) on granular particles of either bioactive glass or wollastonite. For comparative purposes, no bioactive system was used in some tests. Three different methods were used for the immersion of the samples in SBF: 1) 21 days in SBF, 2) 21 days in 1.5 SBF, and 3) 7 days in SBF followed by 14 days in 1.5 SBF (re-immersion method). A bonelike apatite layer was formed on all the samples placed on wollastonite and bioactive glass particles. The morphology of the apatite layer formed by using the re-immersion method and wollastonite closely resembled the existing bioactive systems. No apatite layer was observed on the samples treated without bioactive material and soaked for 21 days in SBF or 1.5 SBF, apart from the substrates treated by using the re-immersion method. The heat treatment delayed the apatite formation in all the cases studied.     

Formation of apatite layers on modified canasite glass-ceramics in simulated body fluid.

Canasite glass-ceramics were modified by either increasing the concentration of calcium in the glass, or by the addition of P2O5. Samples of these novel materials were placed in simulated body fluid (SBF), along with a control material (commercial canasite), for periods ranging from 12 h to 28 days. After immersion, surface analysis was performed using thin film X-ray diffraction, Fourier transform infrared reflection spectroscopy, and scanning electron microscopy equipped with energy dispersive X-ray detectors. The concentrations of sodium, potassium, calcium, silicon, and phosphorus in the SBF solution were measured using inductively coupled plasma emission spectroscopy. No apatite was detected on the surface of commercial canasite, even after 28 days of immersion in SBF. A crystalline apatite layer was formed on the surface of a P2O5-containing canasite after 5 days, and after 3 days for calcium-enriched canasite. Ion release data suggested that the mechanism for apatite deposition was different for P2O5 and non-P2O5-containing glass-ceramics.     

Effect of polyacrylic acid on the apatite formation of a bioactive ceramic in a simulated body fluid: fundamental examination of the possibility of obtaining bioactive glass-ionomer cements for orthopaedic use

Glass-ionomer cements, which consist of CaO-Al2O3-SiO2-CaF2 glass powders and a polyalkenoic acid solution, such as polyacrylic acid (PAA), have been widely used in dentistry. They set rapidly without any shrinkage, the lack of temperature increase on reaction, and develop high mechanical strength. Therefore, if bioactive glass-ionomer cements can be obtained, such cements are expected to be useful as cements for fixing orthopaedic implants to the surrounding bone. In the present study, to examine the possibility of obtaining bioactive glass-ionomer cements, the effect of PAA on the apatite formation on bioactive ceramics in a simulated body fluid was investigated. It was revealed that presence of even a small quantity of PAA inhibits the apatite formation in the body environment. It is speculated that when glass-ionomer cements are implanted into the body, PAA can be released from the glass-ionomer cements and inhibits the apatite formation on their surfaces. It is reasonable to suppose that this will occur with any glass-ionomer cement that contains PAA. Therefore, it might be considered difficult to obtain bioactive glass-ionomer cements.     

Evaluation of apatite ceramics containing alpha-tricalcium phosphate by immersion in simulated body fluid

The purpose of this study was to estimate the availability of alpha-tricalcium phosphate (alpha-TCP) on/in hydroxyapatite (HAP) ceramics for bioactivity as bone-substitute materials by immersion in a simulated body fluid (SBF; Hanks' solution) containing ion concentrations similar to those in human blood plasma. Two alpha-TCP-surface-modified HAP and alpha-TCP-HAP composite materials were prepared by orthophosphoric acid treatment of sintered HAP and controlling the crystal phases of calcium phosphate cement, respectively. After immersion in SBF, the sintered HAP modified on the surface in an approximately 0.2 microm alpha-TCP layer was more effective for the precipitation of carbonated apatites than an approximately 2 microm alpha-TCP layer and HAP-only layer. In the calcium phosphate cements consisting of HAP and alpha-TCP phases, after immersion for 1 week, the specimens precipitated large amounts of apatites having alpha-TCP contents of approximately 25% and 50% in the cement. The results of immersion tests imply the possibility that the alpha-TCP on/in HAP ceramics may be a bioactive agent for bone-substituting HAP materials.     

Bioactivity of tape cast and sintered bioactive glass-ceramic in simulated body fluid

A common ceramic processing technique, tape casting, was used to produce thin, flexible sheets of bioactive glass (Bioglass 45S5) particulate in an organic matrix. Tape casting offers the possibility of producing three-dimensional shapes, as the final material is built up layer by layer. Bioactive glass tapes were sintered together to form small discs for in vitro bioactivity testing in simulated body fluid (SBF). Four different sintering schedules were investigated: 800, 900, and 1000 degrees C for 3 h; and 1000 degrees C for 6 h. Each schedule produced a crystalline material of major phase Na2Ca2Si3O9. Tape cast and sintered bioactive glass-ceramic processed at 1000 degrees C formed crystalline hydroxyapatite layers after 20-24 h in SBF as indicated by Fourier transform infrared spectroscopy, Scanning electron microscopy, and EDS data. FTIR revealed that the greatest amount of hydroxyapatite formation after 2 h was observed for samples sintered at 900 degrees C. The differences in bioactive response were likely caused by the variation in the extent of sintering and, consequently, the amount of surface area available for reaction with SBF.     

An X-ray photoelectron spectroscopy study of the process of apatite formation on bioactive titanium metal

Bioactive titanium metal, prepared by treatment with NaOH followed by an annealing stage to form a sodium titanate layer with a graded structure on its surface, forms a biologically active bone-like apatite layer on its surface in the body, and bonds to bone through this apatite layer. In this study, process of apatite formation on the bioactive titanium metal in a simulated body fluid was investigated using X-ray photoelectron spectroscopy. The bioactive titanium metal formed Ti-OH groups soon after soaking in the simulated body fluid, via the exchange of the Na(+) ions in the sodium titanate on its surface with H(3)O(+) ions in the fluid. The Ti-OH groups on the metal combined with the calcium ions in the fluid immediately to form a calcium titanate. After a long period, the calcium titanate on the metal took the phosphate ions as well as the calcium ions in the fluid to form the apatite nuclei. The apatite nuclei then proceeded to grow by consuming the calcium and phosphate ions in the fluid. These results indicate that the Ti-OH groups formed on the metal induce the apatite nucleation indirectly, by forming a calcium titanate. The initial formation mechanism of the calcium titanate may be attributable to the electrostatic interaction of the negatively charged Ti-OH groups with the positively charged calcium ions.     

Growth of a bonelike apatite on chitosan microparticles after a calcium silicate treatment

Bioactive chitosan microparticles can be prepared successfully by treating them with a calcium silicate solution and then subsequently soaking them in simulated body fluid (SBF). Such a combination enables the development of bioactive microparticles that can be used for several applications in the medical field, including injectable biomaterial systems and tissue engineering carrier systems. Chitosan microparticles, 0.6mum in average size, were soaked either for 12h in fresh calcium silicate solution (condition I) or for 1h in calcium silicate solution that had been aged for 24h before use (condition II). Afterwards, they were dried in air at 60 degrees C for 24h. The samples were then soaked in SBF for 1, 3 and 7 days. After the condition I calcium silicate treatment and the subsequent soaking in SBF, the microparticles formed a dense apatite layer after only 7 days of immersion, which is believed to be due to the formation of silanol (Si-OH) groups effective for apatite formation. For condition II, the microparticles successfully formed an apatite layer on their surfaces in SBF within only 1 day of immersion.     

Simple surface modification of poly(epsilon-caprolactone) for apatite deposition from simulated body fluid

Poly(epsilon-caprolactone) (PCL) with a bone-like apatite layer bound to its surface could be useful as a scaffold for tissue engineering applications. In the present study, the surface of PCL was treated with aqueous NaOH to introduce carboxylate groups onto the surface. The NaOH-treated material was subsequently dipped in aqueous CaCl(2) and K(2)HPO(4).3H(2)O alternately three times to deposit apatite nuclei on the surface. The surface-modified material successfully formed a dense and uniform bone-like surface apatite layer after incubation for 24 h in simulated body fluid with ion concentrations approximately equal to those of human blood plasma.     

Synergistic effect of silanol group and calcium ion in chitosan membrane on apatite forming ability in simulated body fluid

A chitosan membrane modified with silanol groups and calcium ions on its surface and in its structure, respectively, was newly developed and evaluated for the potential application as a bioactive-guided bone-regeneration membrane. The chitosan membrane, which contained calcium nitrate tetrahydrate, was prepared and further subjected to surface modification with 3-isocyanatopropyl triethoxysilane (IPTS) following hydrolysis with HCl solution. As control, chitosan membranes which contained only calcium nitrate tetrahydrate and modified with only silanol groups were prepared, respectively. Three membranes were exposed to simulated body fluid (SBF) for a period ranging from 3 h to 7 days. The SBF exposure led to the deposition of a layer of apatite crystals on the surface of the chitosan membrane modified with silanol groups and calcium ions, while those modified with only calcium ions or silanol groups did not show the apatite-forming ability. It implies that the silanol groups and calcium ion acted together in a synergistic fashion in the formation of apatite crystals; the silanol groups and calcium ions acted as the nucleation sites and accelerator for the formation of apatite crystals, respectively. Therefore, this new chitosan membrane is likely to have a potential for the application as a bioactive guided bone regeneration membrane because of its apatite-forming ability in the SBF.     

Mechanism of bone-like formation on a bioactive implant in vivo

The physical and chemical nature of the remodelled interface between the porous A3 glass-ceramic, composed of (wt%): SiO(2) = 54.5; CaO = 15.0; Na(2)O = 12.0; MgO = 8.5; P(2)O(5) = 6.0 K(2)O = 4.0, and the surrounding bone was studied after implantation into rat tibias. The interfaces which developed new bone layer in direct contact with the implants were examined by analytical scanning and transmission electron microscopy after implantation for 6, 8 and 12 weeks. Degradation processes of the implants also encouraged osseous tissue ingrowths into the pores of the material, changing drastically the macro- and microstructure of the implants. The ionic exchange initiated at the implant interface with the physiological environment was essential in the integration process of the implant, through a dissolution-precipitation-transformation mechanism. The interfaces developed non-toxic biological and chemical activities and remained reactive over the 12-week implantation period. These findings were significant as indicative of morphological and chemical integration of the A3 glass-ceramic into the structure of living bone tissue. A3 glass-ceramic could be suitable for the repair or replacement of living bone.     

Synthesis of bioactive HEMA-MPS-CaCl2 hybrid gels: effects of catalysts in the sol-gel processing on mechanical properties and in vitro hydroxyapatite formation in a simulated body fluid

We investigated synthetic conditions for the fabrication of bioactive hybrid gels from monomers of 2-hydroxyethylmethacrylate (HEMA) and 3-methacryloxypropyltrimethoxysilane (MPS) in combination with CaCl(2), at a starting molar ratio of HEMA: MPS : CaCl(2) of 9 : 1 : 1. Hydroxyapatite formation, essential to show bone bonding, was observed on the HEMA- MPS-CaCl( 2) hybrid gels with the added catalysts NH(3) or HCl with a molar ratio to MPS of 0.1, but not on the hybrid gel with HCl at a molar ratio to MPS of 1. The mechanical properties of the gels were dependent on the catalysts, which may affect the microstructures that develop during sol-gel processing.     

Accelerated bonelike apatite growth on porous polymer/ceramic composite scaffolds in vitro

Although biodegradable polymer/ceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes, the osteogenic potential of these scaffolds needs to be further enhanced for efficient bone tissue engineering. In this study, bonelike apatite was efficiently coated onto the scaffold surface by using polymer/ceramic composite scaffolds instead of polymer scaffolds and by using an accelerated biomimetic process to enhance the osteogenic potential of the scaffold. The creation of bonelike, apatite-coated polymer scaffold was achieved by incubating the scaffolds in simulated body fluid (SBF). The apatite growth on porous poly(D,L-lactic-co-glycolic acid)/nanohydroxyapatite (PLGA/ HA) composite scaffolds was significantly faster than on porous PLGA scaffolds. In addition, the distribution of coated apatite was more uniform on PLGA/HA scaffolds than on PLGA scaffolds. After a 5-day incubation period, the mass of apatite coated onto PLGA/HA scaffolds incubated in 5 x SBF was 2.3-fold higher than PLGA/HA scaffolds incubated in 1 x SBF. Furthermore, when the scaffolds were incubated in 5 x SBF for 5 days, the mass of apatite coated onto PLGA/HA scaffolds was 4.5-fold higher than PLGA scaffolds. These results indicate that the biomimetic apatite coating can be accelerated by using a polymer/ceramic composite scaffold and concentrated SBF. When seeded with osteoblasts, the apatite-coated PLGA/HA scaffolds exhibited significantly higher cell growth, alkaline phosphatase activity, and mineralization in vitro compared to the apatite-coated PLGA scaffolds. Therefore, the apatite-coated PLGA/HA scaffolds may provide enhanced osteogenic potential when used as scaffold for bone tissue engineering.