Kokubo's Simulated Body Fluid Research Articles

The apatite-forming ability of bioactive glasses – A comparative study in human serum and Kokubo's simulated body fluid

Currently, the apatite-forming ability evaluation of a biomaterial employs Kokubo's simulated body fluid (SBF) as a testing medium. As the conventional SBF simulates only the ionic composition and pH of human serum (HS), this study aimed to compare the apatite formation in SBF and HS on the sol-gel-derived bioglass (BG) 83S2 and commercial 45S5 as well as to verify the influence of preincubation of BG in both fluids on cytocompatibility towards human fetal osteoblast cell line hFOB 1.19. The apatite formation in HS was hindered, regardless of the type of used BG. Relatively higher apatite-forming ability was noticed for BG83S2. In direct contact test, both BGs were cytocompatible towards osteoblasts, regardless of the type of used fluid. In the case of extracts; however, only the extract obtained from BG83S2 preincubated in HS did not show the cytotoxic effect. The evaluation of apatite-forming ability in SBF appears to be a fast screening method for comparing the apatite-forming ability of various bioglasses. Nonetheless, SBF is unable to precisely imitate the morphology and crystallinity of apatite observed during the assay in HS, as well as it does not mimic the changes in profiles of osteogenic ions.

Tailoring mechanical and in-vitro cytotoxicity behavior of LZ4X(X=0,1,2&3) magnesium alloy for bone implant application

This present research is focused on understanding the mechanical and bio-corrosion behavior of thermo-mechanically processed i.e. rolled Mg–4Li-XZn alloys with a 90% reduction in thickness. A single-phase Mg–4Li alloy was rolled with a 90% reduction in thickness, reducing the grain size to30 μm, incorporating varying amounts of zinc. Zinc addition and thermo-mechanical processing (hot rolling) at 300 °C refine the grains and increase tensile strength, resulting in LZ43 Mg alloy with an ultimate tensile strength (UTS) of 277 ± 8 MPa and an elongation (El) of 14 ± 3%. Kokubo's simulated body fluid (SBF) was used to perform bio-corrosion and electrochemical tests (PDP and PEIS). SEM, XRD, and FTIR were used to characterize corrosion morphology and bio-mineralization products like hydroxyapatite (HAp) compound. The in-invo corrosion conduction in HEK-293 (Human embryonic kidney cell line) proves that cells were less toxic and 95% of cells remained healthy when cultured in LZ43-HR90 Mg alloy condition.

Enhanced bone repair by silver-doped magnesium calcium phosphate bone cements

The aim was to create magnesium calcium phosphate bone cements (MCPCs) based on struvite and newberyite phases and having antibacterial properties due to the presence of Ag+ ions. An increase of Ag+ concentration from 0.5% to 1.0% substantially enhanced mechanical properties of the cements [compressive strength reached 41 ± 2 MPa (mean ± SD)] and increased the inhibition zone up to 8 mm in a disk diffusion test against Escherichia coli and Staphylococcus aureus without toxicity to MG-63 cells in vitro. Analyses of the MCPC samples soaked in Kokubo's simulated body fluid showed the formation of coatings on the cements' surface, corresponding to a merrillite phase and, after 28 days of the soaking, to a bobierrite phase. Histological examination and micro-CT analysis revealed that the newly developed MCPCs are osteoconductive and biodegradable and may be used as a bone substitute.

Carbon Nanotube (CNT) Encapsulated Magnesium-Based Nanocomposites to Improve Mechanical, Degradation and Antibacterial Performances for Biomedical Device Applications

Nowadays, magnesium (Mg) composites are gaining much attention in biomedical device applications due to their biocompatibility and biodegradability properties. This research is to study the microstructure, mechanical, corrosive and antibacterial properties of Mg−2.5Zn−0.5Zr/xCNT (x = 0, 0.3, 0.6, 0.9) composites made with mechanical alloying and semi-powder metallurgy (SPM) processes, accompanied by SPS. Based on the microstructural characteristics, CNTs were almost uniformly distributed in the Mg matrix. The results displayed that the hardness and ultimate compressive strength (UCS) of the composites were meaningfully increased compared to a Mg matrix. Moreover, the degradation rate of Mg composites was almost halved in the presence of small amounts of CNTs in the Kokubo simulated body fluid (SBF). Due to the slowed degradation process, the Mg−2.5Zn−0.5Zr/0.6CNT biocomposites exhibited excellent cellular compatibility. Evaluation of antibacterial activity displayed that adding CNTs to the Mg matrix could significantly prevent the growing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In general, the research results showed that CNTs are an efficient reinforcement for Mg−2.5Zn−0.5Zr/CNTs biocomposites, which leads to improved mechanical, degradation and antibacterial performances.

Investigation of the wear behavior in simulated body fluid of 316L stainless steels produced by mechanical alloying method

In the present study, the wear behavior of 316L stainless steels (SS) produced with mechanical alloying (MA) method was investigated in simulated body fluid. Alloys produced in different MA times were sintered for 2 h at 1300 °C temperature. Specimens prepared with standard metallographic procedures after sintering, scanning electron microscopy, X-ray diffraction, powder size analyses, hardness, and density measurements were performed. Wear tests were carried out in pin-on-disc type wear device, Kokubo simulated body fluid and body temperature environment (in vitro). Surface roughness was measured before and after the wear test under 40 N at four different sliding distances with 1 ms−1 sliding speed. Results showed that the powder size decreased as the milling time increased and the lowest powder size was measured for the powder milled for 120 min. The highest hardness and lowest weight loss were achieved in specimens milled for 60 min, while the highest average surface roughness was achieved in specimens milled for 30 min.

The Effect of Zirconium Addition on Corrosion Behavior of Zn-Zr Alloys as Biodegradable Orthopedic Implant Application

In this work, corrosion behaviour of zinc-based alloys with addition of 0.5%, 1%, and 2% of zirconium for biodegradable material as orthopaedic implant were investigated. The potentiodynamic polarization method is carried out to determine the corrosion resistance and corrosion rate of each composition in order to observe the effect of zirconium addition in a Kokubo simulated body fluid solution. The result showed that the addition of 0.5% and 1% of zirconium would decrease the corrosion rate of Zn-xZr alloys corresponding to 0.079 mm/year and 0.116 mm/year whereas the 2% addition would increase the rate to 0.188 mm/year due to the formation of Zr-rich precipitates inside the alloys. The passivation zone on the polarization curve showed the formation of the protected thin layer on the surface of the alloys which caused the corrosion rate to decrease, therefore confirmed the degradable ability of the Zn-xZr alloys. In general, the corrosion rates of Zn-xZr alloys were higher than Fe-based alloys and lower than Mg-based alloys. Moreover, the corrosion rates were much lower than the maximum rate of 0.4 mm/year for biodegradable implants so Zn-xZr alloys were suitable as biodegradable material implant for orthopaedic application.

Effects of the Addition of Micro-amounts of Calcium on the Corrosion Resistance of Mg-0.1Mn-1.0Zn-xCa Biomaterials

Magnesium alloys have good biocompatibility and degradability, which are considered ideal characteristics of biomedical implants. However, there are few studies on the influence of the addition of micro-amounts of Ca on magnesium alloys. In this work, the effects of adding micro-amounts of Ca on the microstructure, mechanical properties, and corrosion properties of Mg-0.1Mn-1.0Zn-xCa (x = 0.1, 0.2, and 0.3 wt.%) alloys were investigated. The results reveal that the grain size of the alloy decreased and the yield strength and elongation both increased with increasing Ca concentrations. Corrosion tests in Kokubo simulated body fluid showed that the in vitro immersion rate first slowed down and then increased (6.395 → 6.283 → 6.395 mm/year) with increasing Ca concentrations. This phenomenon was due to the addition of Ca causing grain refinement, which slowed the corrosion rate and generated an Mg2Ca phase to accelerate corrosion. The second-phase Mg2Ca precipitated at the grain boundary and acted as a cathode, and the metal magnesium phase acted as an anode to form a primary battery, which caused corrosion of the magnesium phase and accelerated the corrosion rate.

The fabrication of nanostructured titania polymorphs layer with high crystallinity and its apatite-forming ability

High crystallinity anatase/rutile layers are prepared on the surface of commercially pure titanium (cpTi) by hydrothermal method at 160 °C for 3 h. The surface morphology of titania layers is changed by adjusting the pH value and Ti4+concentration of treating solution (TS). Obtained nanorods on all TS samples are composed of pure rutile and the anatase co-deposited with rutile is found in the dense bottom layer. The possible co-deposition mechanism of polymorphs layers is given in this study. In this study, the rutile nanorods array with high energy (101) facet exposed is found on the TS3 sample. In vitro apatite forming ability of the TS samples are confirmed by soaking them in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5 °C) for 1 and 3 days. Results show that apatite particles could be obtained on all TS samples within 1 d. After 3 days' immersion in SBF, TS3 sample shows strongest apatite X-ray diffraction. These results indicate that the ability in inducing apatite on cpTi with TS3 treatment is excellent. Such excellent apatite-forming ability is ascribed to the existence of thick titania layer with high energy (101) facet exposed on the TS3 sample.

Facile synthesis of novel fibrous silica@apatite@Au composites with superior photo-catalytic activity

Novel fibrous silica@apatite@Au composites with superior photo-catalytic activity were facilely synthesized through combination of electrospinning technique, bio-mineralization, and in situ immobilization. Silica nanofibers with the diameter of 486±60nm were synthesized through electrospinning of the sol-gel mixture of gelatin, tetraethoxysilane, acetic acid, water, and calcium chloride followed by sintering process. After bio-mineralization in the Kokubo's simulated body fluid, silica nanofibers supported in situ deposition of fibrous apatite nanosheet flowers to produce fibrous silica@apatite composites. SEM observations showed that the silica@apatite composites had the fibrous feature with the diameter of 2498±608nm and their surface was very rough and constructed by numerous apatite nanosheets with the thickness around 10nm. XRD patterns showed that the fibrous silica@apatite composites presented the characteristic diffraction peaks at 26° and 32° assigned to apatite components. FT-IR spectra revealed that silica@apatite composites contained both SiOSi bonds and POP groups. After immersion in the suspension of Au nanoparticles, the silica@apatite composites supported the in situ immobilization of Au nanoparticles to produce silica@apatite@Au composites. When incubated in the aqueous mixture of 4-nitrophenol and NaHB4, the fibrous silica@apatite@Au composites presented a superior photo-catalytic activity and rapidly catalyzed the reduction of 4-nitrophenol to 4-aminophenol.

Self-Toughness Porous Sodium Titanate Bioceramics Prepared via In Situ Grown Na2Ti6O13 Rods

Here in, we report the porous bioceramic with Na2Ti6O13 rods prepared by in‒situ growth method. The samples were prepared using cold uniaxial pressing (40 MPa) technique and further sintered at different temperatures. The structure and morphology were characterized by XRD and SEM. The porosity, compressive strength and fracture toughness were also investigated. The bone-like apatite deposition ability of the fabricated ceramic samples was evaluated by Kokubo simulated body fluid (SBF) soaking method. The results indicated that the Na2Ti6O13 rods with about 1‒3 μm in diameter are uniformly distributed in the self‒toughness porous sodium titanate ceramics (SPSTC). The SPSTC with a porosity of 61.10±1.12 % exhibits good compressive strength (43.36±2.43 MPa) and fracture toughness (3.47±0.21 MPa·m1/2). The results indicate that the novel SPSTC scaffolds are promising for bone tissue engineering applications.

Role of electrolyte additives on in-vitro corrosion behavior of DC plasma electrolytic oxidization coatings formed on Cp-Ti

Abstract The present investigation reports the effect of different electrolyte additives on in-vitro corrosion behavior of the plasma electrolytic oxidation (PEO) coatings formed on commercially pure titanium (Cp-Ti). PEO coatings were produced using a pulsed DC power source at a constant current density of 70 mA/cm 2 and a treatment time of 8 min. Different coatings were developed with different additives (sodium sulfate, Tris, di-ammonium hydrogen phosphate and ammonium acetate) added to a base electrolyte consisting of TSOP and KOH. PEO coatings were characterized for phase composition, elemental composition, surface and cross-sectional microstructure, corrosion behavior, apatite forming ability and osteoblast cell adhesion. The corrosion resistance of the developed oxide coatings was evaluated in a Kokubo simulated body fluid (SBF) by potentiodynamic polarization test and EIS study. The in-vitro bioactivity of all the PEO coatings and the untreated substrate was studied by soaking the samples in an SBF environment for 21 days. The TiO 2 layer formed in phosphate, sulfate and nitrite containing electrolyte highly enhances the apatite formation in SBF. An in-vitro cell adhesion test was performed on all coatings using human osteosarcoma cells for 48 h. SEM images of cell adhesion test showed that human osteosarcoma cells were well adhered and spread on all PEO treated samples. Among all the additives, Tris has provided higher corrosion resistance in the SBF environment with good apatite forming ability and osteoblast cell adhesion.

Effects of Alkaline Treatment on Corrosion Behavior of Biodegradable Magnesium

Corrosion behavior of Magnesium substrate were investigated after NaOH treatment in different concentrations (1, 5, and 10 Molar) and duration of (10 min, 30 min, 1 h, 3 h, 6 h and 24 h) at room temperature to be utilized in biomaterial application. Creation of Mg(OH)2 barrier layer after treatment enhanced corrosion resistance. X-Ray diffraction (XRD) was conducted to detect the existence of barrier layer. Potentiodynamic polarization and immersion tests were evaluated the corrosion behavior of Magnesium in Kokubo simulated body fluid (SBF). It is found that Magnesium treated by 1M NaOH for 30 min reveals higher corrosion resistance. In addition this investigation indicates that pH value of Kokubo SBF, strongly influenced by different time and concentration of alkaline treatment

Calcium phosphate crystallization on titania in a flowing Kokubo solution.

Dry titania layers on air-oxidized titanium substrates have been found to be active enough to cause apatite to be deposited in Kokubo's simulated body fluid (SBF) in narrow confined spaces, such as those in narrow grooves and thin gaps. Such in vitro apatite deposition is the basis of the GRAPE(®) technique. The aim of the present study is to determine why GRAPE conditions favor apatite deposition when laminar SBF flow (at 0.01-0.3ml/min) passes through a shallow channel (0.5mm) between a pair of titanium substrates each with a dry layer of titania. Assessing the factors that control the heterogeneous nucleation process led to the proposal of the working hypothesis that there are nucleation pre-embryos, ion assemblies that can be stabilized to form embryos, on the titania layer but that they are removed by the SBF flow. Specimens were subjected to different combinations of processes. One combination was that titania layers were exposed to still or flowing SBF, and the other was that half of a specimen, the inlet or outlet side, was exposed to still or flowing SBF with the other half being covered. The surface morphologies of the specimens were then compared in detail. The conclusion was that exposure to still SBF for 2days before exposure to flowing SBF was required for apatite to be deposited. Some complicated apatite deposition modes were observed, e.g., apatite was deposited even on areas unexposed to still SBF. All of the results were successfully interpreted using the working hypothesis. The conclusion was that the GRAPE(®) technique depends on the confined space holding pre-embryo and embryo assemblies.

Properties of the Ti Void Metal Composites Prepared with Saccharose as a Space Holder and Coated by Hydroxyapatite

In this paper hydroxyapatite biomimetic deposition on Ti void metal composites (Ti-VMC) have been shown. The Ti-VMC were prepared using saccharose (sugar crystals) as a space holder material that forms voids around the Ti scaffold. The Ti (100 and 325 mesh) with sugar (about 0.7−0.9 mm) particles were mixed together with different Ti/sugar ratio and uniaxially pressed. Then the sugar particles were dissolved in water, leaving mechanically bonded Ti particles, forming a metal scaffold. The titanium scaffolds were sintered at 1300°C, which lead to formation the Ti-VMC with voids of diameter of up to 0.9 mm. Because different Ti/saccharose ratio was applied, the Ti-VMC have been made with 50, 60 and 70% porosity. On the as-prepared Ti-VMC the hydroxyapatite (HA) was deposited using mineralisation procedure in Kokubo SBF (simulated body fluid). The Ti-VMC were kept in SBF for time from 7 up to 28 days. Finally the scaffolds were covered by layer of HA showing good corrosion resistance. The mechanical tests show, that most optimal property for implant applications have samples of 50% porosity, made from 100 mesh Ti. The scaffold of 50% porosity states a good background for implant applications.

In vitro apatite formation on nano-crystalline titania layer aligned parallel to Ti6Al4V alloy substrates with sub-millimeter gap.

Pure titanium substrates were chemically oxidized with H2O2 and subsequent thermally oxidized at 400°C in air to form anatase-type titania layer on their surface. The chemically and thermally oxidized titanium substrate (CHT) was aligned parallel to the counter specimen such as commercially pure titanium (cpTi), titanium alloy (Ti6Al4V) popularly used as implant materials or Al substrate with 0.3-mm gap. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5°C) for 7days. XRD and SEM analysis showed that the in vitro apatite-forming ability of the contact surface of the CHT specimen decreased in the order: cpTi>Ti6Al4V>Al. EDX and XPS surface analysis showed that aluminum species were present on the contact surface of the CHT specimen aligned parallel to the counter specimen such as Ti6Al4V and Al. This result indicated that Ti6Al4V or Al specimens released the aluminum species into the SBF under the spatial gap. The released aluminum species might be positively or negatively charged in the SBF and thus can interact with calcium or phosphate species as well as titania layer, causing the suppression of the primary heterogeneous nucleation and growth of apatite on the contact surface of the CHT specimen under the spatial gap. The diffusion and adsorption of aluminum species derived from the half-sized counter specimen under the spatial gap resulted in two dimensionally area-selective deposition of apatite particles on the contact surfaces of the CHT specimen.

Liquid phase deposited titania coating to enable in vitro apatite formation on Ti6Al4V alloy

A recently developed "GRAPE(®) technology" provides titanium or titanium alloy implants with spontaneous apatite-forming ability in vitro, which requires properly designed gaps and optimum heat treatment in air. In this study, titanium alloy and commercially pure (cp) titanium substrates were thermally oxidized in air before aligning pairs of specimens in the GRAPE(®) set-up, i.e., titanium alloy and cp titanium substrates were aligned parallel to each other with optimum gap width (spatial design). A liquid phase deposition (LPD) technique was employed for titania coatings on titanium alloy substrate. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5°C) for 7days to confirm the in vitro apatite formation on the substrates under the specific spatial design. Anatase-type titania coatings fabricated by using LPD technique led to the deposition of apatite particles within 7days and showed apatite X-ray diffraction. On the other hand, thermally oxidized titanium alloy substrate in air and non-treated specimens did not show any apatite X-ray diffraction. These results indicated that the heterogeneous nucleation of apatite induced on anatase-type titania coating prepared by LPD technique when it was aligned parallel to thermally oxidized cp titanium substrate with optimum gap width.

Investigation of silver impact on hydroxyapatite/lignin coatings electrodeposited on titanium

Silver doped hydroxyapatite (HAP) [Ca9.95Ag0.05(PO4)6(OH)2] composite coatings with natural polymer organosolv lignin (Lig) were produced by electrophoretic deposition (EPD) on titanium. Coatings were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), field emission scanning electron microscopy (FE-SEM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The corrosion stability of electrodeposited coatings was evaluated in vitro in Kokubo's simulated body fluid (SBF) at 37 °C using electrochemical impedance spectroscopy (EIS). Antimicrobial properties are directly proportional to the rate of silver ions release from the coatings, determined from inductively coupled plasma spectrometry (ICP-AES). The obtained results are in good agreement with viability of pathogenic bacteria strain Staphylococcus aureus TL in suspension, which had completely disappeared after 24 h. Composite Ag/HAP/Lig coatings were confirmed as non-toxic for healthy immunocompetent peripheral blood mononuclear cells (PBMC).

A Novel Silicon-Based Electrochemical Treatment to Improve Osteointegration of Titanium Implants

Titanium and its alloy represent the most commonly used biomaterials worldwide designed for bone-contact under-load applications, which often require specific mechanical properties. In particular, a large number of different biomimetic surface treatments have been developed to speed up the osteointegration process, which facilitates a reduction in recovery time. The aim of this work is to investigate the physical-chemical, mechanical and bioactivity properties of an innovative biomimetic treatment on titanium performed using Anodic Spark Deposition (ASD) electrochemical treatment. The proposed ASD treatment was obtained in an electrochemical solution containing silicon, calcium, phosphorous and sodium followed by an alkali etching. Surface morphology was characterized using SEM and laser profilometry. Chemical and structural composition was assessed by EDS, ICP/OES and XRD analysis. Vickers micro hardness and static contact angle measurements were performed to assess the surface mechanical properties and wettability. The proposed anodization treatment was capable of providing a chemical and morphologic modified titanium oxide layer, adherent and characterized by superhydrophilic properties. The microporous morphology was enriched by calcium, silicon, sodium and phosphorous.After incubation in Kokubo's Simulated Body Fluid (SBF) the treatment showed very high mineralization potential compared to the reference surfaces, accounting for a deposited hydroxyapatite layer as thick as 12 μm after 14 days of SBF incubation. On the basis of the results obtained in this study, we believe that the novel silicon-based ASD biomimetic treatment represents a promising treatment capable of enhancing the osteointegration of titanium for dental and orthopedic applications.

Bioactivity of Calcium Aluminate Endodontic Cement

IntroductionCalcium aluminate endodontic cement (CAEC) developed for use in root canal therapy has been produced with additives that improve handling properties and provide higher mechanical strength than mineral trioxide aggregate (MTA) according to prior studies. The aim of this study was to evaluate the bioactivity of CAEC containing accelerating additives (A-CAEC) in comparison with MTA, both in contact with simulated body fluid (SBF) solutions. MethodspH measurements were taken for set cement samples immersed in water or SBF solutions prepared according to the Kokubo and Rigo techniques. The surface of these materials kept in contact with SBF solutions were also evaluated by means of scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray diffraction. ResultsBecause of the calcium hydroxide dissociation, MTA cement is able to release more Ca2+ ions and results in a higher pH increase compared with A-CAEC. This behavior enhances the supersaturation of Kokubo solution, resulting in the precipitation of calcium phosphate phases on the MTA surface. On the other hand, for MTA in Rigo SBF solution, the pH value attained was higher than for the Kokubo SBF solution as a result of the Mg2+ ion precipitation, which inhibited the calcium phosphate phase formation. For A-CAEC, the optimal precipitation conditions of calcium phosphate phases are achieved in Rigo SBF solution. ConclusionsMTA and A-CAEC present bioactivity in contact with SBF solution although the composition of this solution defines the type of phase precipitated.

Effect of calcium content on the microstructure, hardness and in-vitro corrosion behavior of biodegradable Mg-Ca binary alloy

Effect of calcium addition on microstructure, hardness value and corrosion behavior of five different Mg-xCa binary alloys (x = 0.7, 1, 2, 3, 4 wt. (%)) was investigated. Notable refinement in microstructure of the alloy occurred with increasing calcium content. In addition, more uniform distribution of Mg 2 Ca phase was observed in α-Mg matrix resulted in an increase in hardness value. The in-vitro corrosion examination using Kokubo simulated body fluid showed that the addition of calcium shifted the fluid pH value to a higher level similar to those found in pure commercial Mg. The high pH value amplified the formation and growth of bone-like apatite. Higher percentage of Ca resulted in needle-shaped growth of the apatite. Electrochemical measurements in the same solution revealed that increasing Ca content led to higher corrosion rates due to the formation of more cathodic Mg 2 Ca precipitate in the microstructure. The results therefore suggested that Mg-0.7Ca with the minimum amount of Mg 2 Ca is a good candidate for bio-implant applications.