In vitro bioactivity of a biocomposite fabricated from HA and Ti powders by powder metallurgy method

Traditionally, hydroxyapatite was used as a coating material on titanium substrate by various techniques. In the present work, a biocomposite was successfully fabricated from hydroxyapatite and titanium powders by powder metallurgy method. Bioactivity of the composite in a simulated body fluid (SBF) was investigated. Main crystal phases of the as-fabricated composite are found to be Ti2O, CaTiO3, CaO, alpha-Ti and a TiP-like phase. When the composite is immersed in the simulated body fluid for a certain time, a poor-crystallized, calcium-deficient, carbonate-containing apatite film will form on the surface of the composite. The time required to induce apatite nucleation is within 2 h. In addition, the apatite is also incorporated with a little magnesium and chlorine element. It is found that Ti2O has the ability to induce the formation of bone-like apatite in the SBF. And a dissolve of the CaO phase could also provide favorable conditions for the apatite formation, by forming open pores on the surface of the composite and increasing the degree of supersaturation of the SBF with respect to the apatite.     

Surface modifications of alumina-silica glass fiber

A commercial glass fiber with Al(2)O(3) (68.4%) and SiO(2) (27.6%) as major components and CaO, TiO(2), Fe(2)O(3), and CuO as minor components was used as substrate in a silica sol-gel coating process. After cleaning, fiber samples were immersed into tetraethoxysilane (TEOS) at room temperature for 1 h, and then individual fiber samples were soaked into a simulated body fluid (SBF) solution,1 and removed after 5, 10, 15, and 20 days. Zeta potential and Energy Dispersive Spectroscopy (EDS) analyses showed that the fiber surfaces were effectively coated with a silica layer, which improved the formation of an HA layer upon immersion into SBF solution for 5 days. The coating became even more continuous after 10-day immersion. Fourier Transform Infrared Spectroscopic (FTIR) analyses confirmed that the coating layer has P--O vibration bands characteristic of hydroxyapatite (HA) near 1060 and 600 cm(-1).     

Bioactivity of plasma sprayed dicalcium silicate coatings.

Dicalcium silicate coatings on titanium alloys substrates were prepared by plasma spraying and immersed in simulated body fluids for a period of time to investigate the nucleation and growth of apatite on the surface of the coatings. Surface structural changes of the specimens were analyzed by XRD and IR technologies. SEM and EDS were used to observe surface morphologies and determine the composition of dicalcium silicate coatings before and after immersion in simulated body fluid. The plasma sprayed dicalcium silicate coating was bonding tightly to the substrate. The coating was mainly composed of beta-Ca2SiO4 and glassy phase. A dense carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the plasma sprayed dicalcium silicate coating soaked in SBF solution for 2 days. In addition, a silica-rich layer was also observed between CHA layer and coatings. With an increase in the immersion time, the CHA layer gradually became thicker. The results obtained indicated that the plasma sprayed dicalcium silicate coating possesses excellent bioactivity.     

Activity of plasma sprayed yttria stabilized zirconia reinforced hydroxyapatite/Ti-6Al-4V composite coatings in simulated body fluid

Hydroxyapatite (HA)/yttria stabilized zirconia/Ti-6Al-4V bio-composite coatings deposited onto Ti-6Al-4V substrate through a plasma spray technique were immersed in simulated body fluid (SBF) to investigate their behavior in vitro. Surface morphologies and structural changes in the coatings were analyzed by scanning electron microscopy, thin-film X-ray diffractometer, and X-ray photoelectron spectroscopy. The tensile bond strength of the coatings after immersion was also conducted through the ASTM C-633 standard for thermal sprayed coatings. Results showed that carbonate-containing hydroxyapatite (CHA) layer formed on the surface of composite coatings after 4 weeks immersion in SBF solution, indicating the composite coating possessed excellent bioactivity. The mechanical properties were found to decrease with immersion duration of maximum 56 days. However, minimal variation in mechanical properties was found subsequent to achieving supersaturation of the calcium ions, which was attained with the precipitation of the calcium phosphate layers. The mechanical properties of the composite coating were found to be significantly higher than those of pure HA coatings even after immersion in the SBF solution, indicating the enhanced mechanical properties of the composite coatings.     

Surface characteristics and dissolution behavior of plasma-sprayed hydroxyapatite coating

One of the most important concerns with the clinical use of plasma-sprayed hydroxyapatite (HA) coatings is the resorption of the coating, and dissolution at neutral pH is one of the two major resorption mechanisms. In this study, highly crystalline pure HA powders were atmospherically plasma sprayed using various parameters. Dissolution of both HA powders and coatings was measured using a calcium ion meter. Surface characteristics, including phase, morphology, and roughness, were compared for the coatings before and after dissolution. Pulverized HA coatings exhibited significantly higher dissolution compared with the same quantity of feedstock HA powders because of the decreased crystallinity and fine crystal size of the coating. Furthermore, the dissolution decreased with the crystallinity of the coating. Dissolution of HA coatings did not show much difference with respect to the coatings in the initial stage of immersion (4 h). However, dissolution of all coatings reached saturation in a fresh physiological solution. The saturation values were much lower compared with their counterparts in the form of powders, which may imply the stability of HA coatings in long-term use. In addition to crystallinity, the particle melting status in the coatings, i.e., the volume of nanocrystals, and porosity, was found to be another important factor for the dissolution of the HA coating. X-ray diffraction patterns of HA coatings indicated the complete dissolution of impurity phases and amorphous phase after the coatings were immersed in the solution for 4 days. Coatings sprayed at lower power (27.5 kW) exhibited a pattern of crystalline HA whereas coatings sprayed at higher power (42 kW) exhibited a pattern of bone apatite. Surface morphologies showed preferential dissolution of amorphous phase in all coatings accompanied with precipitation of bone apatite observable for coatings sprayed at higher power. Surface roughness measured after the dissolution studies increased for the two coatings sprayed at lower power level but decreased for coatings sprayed at higher power level. This decrease is attributed to the better match in solubility characteristics between the fine crystals and the amorphous calcium phosphate within the coating.     

Phase composition and in vitro bioactivity of porous implants made of bioactive glass S53P4

This work studied the influence of sintering temperature on the phase composition, compression strength and in vitro properties of implants made of bioactive glass S53P4. The implants were sintered within the temperature range 600-1000°C. Over the whole temperature range studied, consolidation took place mainly via viscous flow sintering, even though there was partial surface crystallization. The mechanical strength of the implants was low but increased with the sintering temperature, from 0.7 MPa at 635°C to 10 MPa at 1000°C. Changes in the composition of simulated body fluid (SBF), the immersion solution, were evaluated by pH measurements and ion analysis using inductively coupled plasma optical emission spectrometry. The development of a calcium phosphate layer on the implant surfaces was verified using scanning electron microscopy-electron-dispersive X-ray analysis. When immersed in SBF, a calcium phosphate layer formed on all the samples, but the structure of this layer was affected by the surface crystalline phases. Hydroxyapatite formed more readily on amorphous and partially crystalline implants containing both primary Na(2)O·CaO·2SiO(2) and secondary Na(2)Ca(4)(PO(4))(2)SiO(4) crystals than on implants containing only primary crystals.     

Immersion behavior of RF magnetron-assisted sputtered hydroxyapatite/titanium coatings in simulated body fluid.

The focus of the present study was on the dissolution/degradation behavior of a series of magnetron-sputtered, single-layered HA/Ti coatings on Ti-6Al-4V substrate immersed in SBF. Changes in coating morphology, crystal structure, and adhesion strength with immersion time are characterized. XRD, FTIR, and LVSEM results consistently indicate that highly crystalline monolithic HA coating is very dissolvable in SBF. The monolithic HA coating is largely delaminated in 3 weeks and entirely peeled off the substrate in 7 weeks. The dissolution is even greater for 95HA/5Ti coating, which severely disintegrated in only 1 week. The amorphous-like coatings sputtered from targets comprising 10 vol % or more Ti, however, appeared almost intact, and their adhesion strengths, which were all higher than 60 MPa, did not change much (within 10%) even after 14 weeks of immersion. The coatings from targets comprising roughly 10-50 vol % Ti combine advantages of high and nondeclining adhesion strength, high resistance to SBF attack, and possibly much higher bioactivity (with large amounts of Ca, P, etc., dissolved in the coatings) than that of Ti.     

Dissolution and mineralization behaviors of HA coatings

The dissolution and mineralization behavior of HA coatings are two of the main factors governing the bioactivity of coatings. After different post treatment operators, the plasma-sprayed HA coatings have different characteristics, including different chemical composition, crystallinity, crystallite size and dissolution behavior. In this study, HA coatings were characterized by X-ray diffraction, scanning electron microscope, and Fourier transform infrared spectra before and after immersion in simulated body fluid (SBF). When immersed in SBF, both dissolution and precipitation occurred at the same time, but the kinetics of dissolution was quite different from that of precipitation. The former was dominated by ion exchange, while the latter was controlled by the ion concentration product and the solubility of the particles. Therefore, the dissolution behaviors of phosphate ions partly depended on the dissolution behaviors of calcium ions. With the increase of ions concentration in solution by dissolution, more nucleation sites appeared on the surface of coatings. Crystalline grains gradually grew up on the nucleation sites and developed into biomineral layers. The biomineral layers were the results of the precipitation of the ions in the solution; and the carbonates partially substituted phosphates to form bone-like apatite. The different dissolution characters resulted in quite different morphology of the biomineral layers: the coatings with low solubility induced biomineral layers of large grains; on the contrary, the biomineral layers of network structure were observed on the more soluble coatings.     

In vitro studies of plasma-sprayed hydroxyapatite/Ti-6Al-4V composite coatings in simulated body fluid (SBF)

The bioactivity of plasma-sprayed hydroxyapatite (HA)/Ti-6Al-4V composite coatings was studied by soaking the coatings in simulated body fluid (SBF) for up to 8 weeks. This investigation was aimed at elucidating the biological behaviour of plasma-sprayed HA/Ti-6Al-4V composite coatings by analyzing the changes in chemistry, and crystallinity of the composite coating in a body-analogous solution. Phase composition, microstructure and calcium ion concentration were analyzed before, and after immersion. The mechanical properties, such as tensile bond strength, microhardness and Young's modulus were appropriately measured. Results demonstrated that the tensile bond strength of the composite coating was significantly higher than that of pure HA coatings even after soaking in the SBF solution over an 8-weeks period. Dissolution of Ca-P phases in SBF was evident after 24h of soaking, and, a layer of carbonate-apatite covered the coating surface after 2 weeks of immersion. The mechanical properties were found to diminish with soaking duration. However, slight variation in mechanical properties was found after supersaturation of the calcium ions was attained with the precipitation of the calcium phosphate layers.     

Microstructure and biological properties of micro-arc oxidation coatings on ZK60 magnesium alloy

Ceramic coatings were prepared on ZK60 magnesium alloy in electrolyte with different concentration ratio of calcium and phosphorus (Ca/P) by micro-arc oxidation (MAO) technique at constant voltage. The microstructure, phase composition, elemental distribution, corrosion resistance, and adhesion of the coatings were investigated by scanning electron microscope (SEM), X-ray diffractometer (XRD), energy-dispersive X-ray spectrometry (EDS), electrochemical workstation, and scratch spectrometer, respectively. The coating biocompatibility was evaluated by in vitro cytotoxicity tests and systemic toxicity tests, and the bioactivity and degradability were evaluated by simulation body fluid (SBF) immersion tests. SEM shows that pores with different shapes distribute all over the coating surface. The adhesion and thickness of the coatings increases with increasing Ca/P ratio of electrolyte. The in vitro cytotoxicity tests and systemic toxicity texts demonstrate that the coatings have no toxicity to cell and living animal, which show that the coatings have excellent biocompatibility. XRD analysis shows that bioactive calciumphosphate (CaP) phases such as hydroxyapatite (HA, Ca(10)(PO(4))(6)(OH)(2)) and calcium pyrophosphate (CPP, Ca(2)P(2)O(7)) are induced in the immersed coatings, indicating that the MAO coatings have excellent bioactivity.     

Bioactive hydroxyapatite coatings on polymer composites for orthopedic implants

Hydroxyapatite [HA, Ca10(PO4)6(OH)2] coatings on polymer composite substrates were investigated for their bioactivity and their physicochemical and mechanical characteristics. HA holds key characteristics for use in orthopedic applications, such as for coating of the femoral stem in a hip replacement device. The plasma-spray technique was used to project HA onto a carbon fiber/polyamide 12 composite substrate. The resulting HA coatings exhibited mechanical adhesion as high as 23 MPa, depending on the surface treatment of the composite substrate. The purpose of this investigation was to evaluate the bioactivity of an HA-coated composite substrate. HA- coated samples have been immersed in simulated body fluid (SBF) and maintained within a shaker bath for periods of 1, 7, 14, 21, and 28 days at 37 degrees C. Scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction techniques were performed on the samples before and after immersion into SBF. SBF was analyzed using inductively coupled plasma atomic emission spectrometry for element concentration and evaluation of the solution's purity. SBF conditioning led to the deposition of crystalline HA onto the surface of the coatings. The calcium-to-phosphorous ratios of initial HA coating and of newly deposited HA were respectively 1.72 and 1.65, close to the HA theoretical calcium/phosphorous value of 1.67. Results demonstrated that bioactive HA coatings were produced by plasma spraying, because SBF conditioning induced newly formed HA with high crystallinity. Mechanical adhesion of the HA coatings was not significantly affected upon SBF conditioning.     

Early apatite deposition and osteoblast growth on plasma-sprayed dicalcium silicate coating

Dicalcium silicate coating was deposited onto a Ti-6Al-4V substrate using plasma-spraying technology. The coating was immersed in simulated body fluid (SBF) for 1, 3, 6, 12, 24, and 48 h to investigate early apatite formation on the coating. Osteoblasts were also seeded onto the surface of the dicalcium silicate coating to evaluate its biocompatibility. Cold field-emission scanning electron microscopy and energy-dispersive X-ray spectrometry were used to evaluate the morphologies and determine the chemical composition of the coatings. The surface structural changes caused by immersion in SBF were analyzed using thin-film X-ray diffraction. After the dicalcium silicate coating was soaked in SBF solution 1-6 h, two types of particles containing calcium and phosphorus were formed on the surface. One type consisted of relatively larger particles (P1) precipitated on the surface of the coating from the precursor cluster formed in the SBF solution. The second type was composed of particles (P2) nucleated on the surface of the coating. With increasing immersion time, the particles coalesced to form a surface Ca-P layer. The Ca-P layer was composed of amorphous calcium phosphate that was not transformed to crystalline apatite until the immersion time in SBF exceeded 24 h. The formation mechanism of the Ca-P layer and apatite on the surface of the coating is believed to be involved in the formation of the Si 3-ring active surface site with negative charge. The cell-seeding test revealed that osteoblasts grew and proliferated very well on the surface of the dicalcium silicate coating.     

磁控溅射制备HA/Ti6Al4V复合材料种植体体外生物活性研究

研究了射频磁控溅射法制备的HA/Ti6Al4V复合材料种植体在模拟体液(Simulated body fluid,SBF)环境下的生物活性。利用扫描电镜(SEM)、能谱分析(EDS)、红外光谱(FTIR)及X-射线衍射(XRD)分析了该种植体涂层在模拟体液中浸泡前后的表面形貌、界面结合状态、晶体结构和相组成的变化,结果表明:该种植体涂层在模拟体液中存在溶解和新生物质在其表面沉积相伴的过程。其中,HA涂层表面的新生物质是一种缺钙型且含有CO32-的类骨磷灰石,其n(Ca)/n(P)比值约为1.56,晶粒小,结晶度低,接近于非晶态,这与自然骨中无机相的结构成分相似,因此具有良好生物相容性和生物活性。     

The influence of the phosphorus content on the bioactivity of sol-gel glass ceramics

The aim of this work was to study the influence of the phosphorus on the crystallization and bioactivity of glass-ceramics obtained from sol-gel glasses. For this purpose two sol-gel glasses with a similar composition but one of them containing P2O5 (70% SiO2; 30% CaO and 70% SiO2; 26% CaO; 4% P2O5, mol%) were prepared. Pieces of these glasses were treated at temperatures ranging between 700 degrees C and 1400 degrees C for 3 h. The obtained materials were characterized by XRD, FTIR, SEM-EDS and the biaxial flexural strength was determined in samples heated at 1100 degrees C. In addition, an in vitro bioactivity study in simulated body fluid (SBF) was carried out. The results showed that phosphorus plays an important role in the crystallization of the glasses: it induced the crystallization of calcium phosphate phases, the stabilization of the wollastonite phase at high temperature as well as the crystallization of SiO2 phases at low temperatures. Moreover, the presence of phosphorus produced a heterogeneous distribution of defects in the pieces and, therefore, the flexural strength of samples containing this element decreased. Finally, glass-ceramics obtained from glasses containing phosphorus showed the fastest formation rate of the apatite layer when soaked in SBF.     

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.     

In vitro behavior of sintered powder injection molded Ti-6Al-4V/HA

This article reports the morphology and mechanical properties of sintered powder injection molded Ti-6Al-4V/HA parts in a simulated physiological environment. Sintered Ti-6Al-4V/HA parts were immersed in a simulated body fluid (SBF) with ion concentrations that were comparable to those of human blood plasma for a total period of 12 weeks. At intervals of 2 weeks, the immersed Ti-6Al-4V/HA parts were analyzed with the use of scanning electron microscopy (SEM), X-ray diffractometry (XRD), and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Mechanical properties such as flexural strength, flexural modulus, compressive strength, and compressive modulus were also evaluated. Results showed that complete dissolution of the more soluble phases such as tricalcium phosphate (TCP), tetracalcium phosphate (TTCP), and calcium oxide (CaO) were found after 2 weeks of immersion in SBF. ICP analysis showed that high calcium concentration release of around 200 ppm was observed in the SBF solution after 2-4 weeks of immersion, indicating that dissolution has taken place. Next, a gradual decrease in calcium concentration release in the SBF solution was observed after immersion for 4-6 weeks, with increasing amounts of calcium phosphate precipitates being observed on the Ti-6Al-4V/HA surface. Mechanical properties such as strength and modulus were found to deteriorate during 2-4 weeks of immersion, followed by gradual increment as the immersion period increased. This study also showed that parts sintered at 1150 C exhibited faster dissolution and precipitation rates than parts sintered at 1050 C in a physiological environment.     

Bone-like apatite layer formation on hydroxyapatite prepared by spark plasma sintering (SPS)

Hydroxyapatite (HA) compacts with high density and superior mechanical properties were fabricated by spark plasma sintering (SPS) using spray-dried HA powders as feedstock. The formation of bone-like apatite layer on SPS consolidated HA compacts were investigated by soaking the HA compacts in simulated body fluid (SBF) for various periods (maximum of 28 days). The structural changes in HA post-SBF were analyzed with scanning electron microscopy, grazing incidence X-ray diffraction and X-ray photoelectron spectroscopy. It was found that a layer consisting microcrystalline carbonate-containing hydroxyapatite was formed on the surface of HA compacts after soaking for 24h. The formation mechanism of apatite on the surface of HA compacts after soaking in SBF was attributed to the ion exchange between HA compacts and the SBF solution. The increase in ionic concentration of calcium and phosphorus as well as the increase in pH after SBF immersion resulted in an increase in ionic activity product of apatite in the solution, and provided a specific surface with a low interface energy that is conducive to the nucleation of apatite on the surface of HA compacts.     

TRIS buffer in simulated body fluid distorts the assessment of glass-ceramic scaffold bioactivity

The paper deals with the characterisation of the bioactive phenomena of glass-ceramic scaffold derived from Bioglass? (containing 77 wt.% of crystalline phases Na(2)O·2CaO·3SiO(2) and CaO·SiO(2) and 23 wt.% of residual glass phase) using simulated body fluid (SBF) buffered with tris-(hydroxymethyl) aminomethane (TRIS). A significant effect of the TRIS buffer on glass-ceramic scaffold dissolution in SBF was detected. To better understand the influence of the buffer, the glass-ceramic scaffold was exposed to a series of in vitro tests using different media as follows: (i) a fresh liquid flow of SBF containing tris (hydroxymethyl) aminomethane; (ii) SBF solution without TRIS buffer; (iii) TRIS buffer alone; and (iv) demineralised water. The in vitro tests were provided under static and dynamic arrangements. SBF buffered with TRIS dissolved both the crystalline and residual glass phases of the scaffold and a crystalline form of hydroxyapatite (HAp) developed on the scaffold surface. In contrast, when TRIS buffer was not present in the solutions only the residual glassy phase dissolved and an amorphous calcium phosphate (Ca-P) phase formed on the scaffold surface. It was confirmed that the TRIS buffer primarily dissolved the crystalline phase of the glass-ceramic, doubled the dissolving rate of the scaffold and moreover supported the formation of crystalline HAp. This significant effect of the buffer TRIS on bioactive glass-ceramic scaffold degradation in SBF has not been demonstrated previously and should be considered when analysing the results of SBF immersion bioactivity tests of such systems.     

Nanocrystalline hydroxyapatite coatings on titanium: a new fast biomimetic method

We obtained a fast biomimetic deposition of hydroxyapatite (HA) coatings on Ti6Al4V substrates using a slightly supersaturated Ca/P solution, with an ionic composition simpler than that of simulated body fluid (SBF). At variance with other fast deposition methods, which produce amorphous calcium phosphate coatings, the new proposed composition allows one to obtain nanocrystalline HA. Soaking in supersaturated Ca/P solution results in the deposition of a uniform coating in a few hours, whereas SBF, or even 1.5SBF, requires 14 days to deposit a homogeneous coating on the same substrates. The coating consists of HA globular aggregates, which exhibit a finer lamellar structure than those deposited from SBF. The extent of deposition increases on increasing the immersion time. Transmission electron microscope (TEM) images recorded on the material detached from the coating show that the deposition is constituted of thin nanocrystals. Electron diffraction (ED) patterns recorded from most of the crystals exhibit the presence of rings, which can be indexed as reflections characteristic of HA. Furthermore, several HA single-crystal spot ED images were obtained from individual crystals.     

Biomimetic apatite coatings on micro-arc oxidized titania

Biomimetic apatite coatings on micro-arc oxidized titania films were investigated and their apatite-inducing ability was evaluated in a simulated body fluid (1.0 SBF) as well as in a 1.5 times concentrated SBF (1.5 SBF). Titania-based films on titanium were prepared by micro-arc oxidation at various applied voltages (250-500 V) in an electrolytic solution containing beta-glycerophosphate disodium salt pentahydrate (beta-GP) and calcium acetate monohydrate (CA). Macro-porous, Ca- and P-containing titania-based films were formed on the titanium substrates. The phase, Ca and P content, morphology, and thickness of the films were strongly dependent on the applied voltage. In particular, Ca- and P-containing compounds such as CaTiO3, beta-Ca2P2O7 and alpha-Ca3(PO4)2 were produced at higher voltages (>450 V). When immersed in 1.0 SBF, a carbonated hydroxyapatite was induced on the surfaces of the films oxidized at higher voltages (>450 V) after 28 days, which is closely related to the Ca- and P-containing phases. The use of 1.5 SBF shortened the apatite induction time and apatite formation was confirmed even on the surface of the films oxidized at 350 V, which suggests that the incorporated Ca and P in the titania films play a similar role to the Ca- and P-containing compounds in the SBF.