Comparison of in vitro and in vivo bioactivity of SrO-CaO-ZnO-SiO2 glass grafts

A range of calcium-strontium-zinc-silicate glass grafts are developed. Following characterization, their ability to form an apatite layer in simulated body fluid (SBF) is evaluated. Concurrently, their in vivo biocompatibility is determined. These glasses are incapable of forming an apatite layer in SBF. However, in vivo, each glass is well tolerated with new bone formation apparent in close apposition to implanted particles and no evidence of an inflammatory response. Such results are contrary to much of the literature and indicate that forecasting a materials ability to bond to bone based on SBF experiments may provide a false negative result.     

Synthesis of osteoconductive organic inorganic nanohybrids through modification of chitin with alkoxysilane and calcium chloride

The so-called bioactive ceramics have been attractive because they spontaneously bond to living bone. Organic-inorganic hybrids consisting of organic polymers and the essential constituents of the bioactive ceramics, i.e., silanol (Si-OH) group and calcium ions (Ca(2+)), are useful as novel bone substitutes, owing to bioactivity and high flexibility. In the present study, organic-inorganic hybrids are synthesized from chitin by modification with glycidoxypropyltrimethoxysilane (GPS) and calcium chloride (CaCl(2)). Their apatite-forming ability is examined in a simulated body fluid (SBF). The prepared hybrids form apatite on their surfaces in SBF within 7 days.     

Push-out testing and histological evaluation of glass reinforced hydroxyapatite composites implanted in the tibia of rabbits.

In vitro and in vivo bioactivity studies were performed to assess the biocompatibility of CaO-P2O5 glass-reinforced hydroxyapatite (GR-HA) composites. The ability to form an apatite layer by soaking in simulated body fluid (SBF) was examined and surfaces were characterized using FTIR reflection and thin-film X-ray diffraction analyses. Qualitative histology, histomorphometric measurements, and push-out testing were performed in a rabbit model for characterizing bone/implant bonding. Under the in vitro conditions using SBF, an apatite layer could not be formed on GR-HA composites within 8 weeks. Results of push-out testing showed bonding between the composites and bone, ranging from 130-145 N after 2 weeks of implantation. After the longest implantation period, 16 weeks, the GR-HA composite prepared with the higher content of CaO-P2O5 glass showed the highest bonding force, 606 +/- 45 N, compared to 459 +/- 30 N for sintered HA. Development of immature bone and modifications in the turnover of a more mature bone on the surface of GR-HA composites were similar to those on sintered HA.     

Enhancing the bioactivity of zirconia and zirconia composites by surface modification

Among bioceramics, zirconia (ZrO(2)) and alumina (Al(2)O(3)) possess exceptional mechanical properties suitable for load-bearing and wear-resistant applications but the poor bioactivity of these materials is the major concern when bonding and integration to the living bone are desired. This article investigates two different approaches and their underlying mechanisms to improve the bioactivity of zirconia (3Y-TZP) and a zirconia composite with alumina (10Ce-TZP/Al(2)O(3)). Chemical treatment approach applied on 3Y-TZP where the substrates were soaked in 5M H(3)PO(4) to create chemically functional groups on the surface for inducing apatite nucleation. X-ray photoelectron spectroscopy (XPS) was used to detect chemical changes and X-ray diffraction (XRD) to monitor phase changes on the surface before and after acid treatment. Alternate soaking approach applied on 10Ce-TZP/Al(2)O(3) consisted of soaking the composite substrates in CaCl(2) and Na(2)HPO(4) solutions alternately to make a precursor for apatite formation. The bioactivity was evaluated by apatite-forming ability of surface-treated materials in simulated body fluid (SBF). Both methods resulted in the formation of hydroxyapatite on the surface of materials; however, alternate soaking approach showed to be a simpler, faster, and more effective method than the chemical treatment approach for enhancing the bioactivity of zirconia materials.     

Formation of CaTiO3/TiO2 composite coating on titanium alloy for biomedical applications

Plasma electrochemical oxidation (PEO) was used to prepare TiO2-based coating containing Ca and P on titanium alloy. After alkali- and then heat-treatment at 800 degrees C of the PEO coating, a CaTiO3/TiO2 composite (CTC) coating was obtained. The current results indicate that the apatite-forming ability of the CTC coating is higher than that of the PEO coating. During the simulated body fluid (SBF) incubation, Ca of the CTC coating is released into the SBF. An ionic exchange between Ca(2+) ions of the CTC coating and H(3)O(+) ions of the SBF may take place during the SBF incubation. As a result, the abundant Ti--OH groups are formed on the surface of the CTC coating. The hydroxyl functionalized surface greatly enhances the nucleation and growth of apatite, leading to the high apatite-forming ability of the CTC coating. The apatite induced by the CTC coating exhibits a porous and carbonated structure.     

In vitro apatite formation and its growth kinetics on hydroxyapatite/polyetheretherketone biocomposites

The formation of biologically equivalent carbonate-containing apatite on the surface of synthetic hydroxyapatite (HA) is an important step leading to good bone healing. In this study, HA-reinforced polyetheretherketone (PEEK) composites were prepared by homogeneous mixing of HA and PEEK powders, compaction, and pressureless sintering. The bioactivity of HA/PEEK composite with 10, 20, 30 and 40 vol% HA was evaluated by immersing the composite disks in the simulated body fluid (SBF) for up to 4 weeks. The surface of composite with 40 vol% HA was covered by a layer of bone-like apatite just after 3 days of immersion, while 10 vol% HA was covered only after 28 days. This apatite layer was characterized by SEM, thin film X-ray diffractometer, attenuated total reflectance-Fourier transform infrared spectrometer (FTIR)/FTIR. Introducing a concept called apatite-forming capacity of SBF, growth kinetics of the apatite layer on the surface of the composite disks was carried out. The growth rate constant increased with HA volume fraction of the composite, suggesting that the bioactivity of the HA/PEEK composite increases with increasing HA volume fraction in the composite.     

How useful is SBF in predicting in vivo bone bioactivity?

The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. However, the validity of this method for evaluating bone-bonding ability has not been assessed systematically. Here, the history of SBF, correlation of the ability of apatite to form on various materials in SBF with their in vivo bone bioactivities, and some examples of the development of novel bioactive materials based on apatite formation in SBF are reviewed. It was concluded that examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material, and the number of animals used in and the duration of animal experiments can be reduced remarkably by using this method.     

Characteristic and in vitro bioactivity of a microarc-oxidized TiO(2)-based coating after chemical treatment

Microarc oxidation (MAO) was used to prepare a TiO(2)-based coating containing Ca and P on titanium alloy. An alkali treatment was developed to modify the surface of the MAO coating to improve the apatite-forming ability of the coating. The chemically treated MAO coating exhibits a modified layer, with the main constituents being O, Ti, Ca and Na, showing anatase. The modified MAO coating shows a rough and porous morphology containing numerous nanoflakes of approximately 100nm thickness. During the alkali treatment process, P on the surface of the MAO coating shows a main dynamic process of dissolution; however, Ca exhibits a re-deposition process as well as dissolution. The formation of the modified layer could be explained by this mechanism: negatively charged HTiO(3)(-) ions are formed on the MAO coating due to the attack of OH(-) ions on the TiO(2) phase. The HTiO(3)(-) ions could incorporate sodium from the alkali solution and calcium from the alkali solution and MAO coating. The apatite-forming ability of the MAO coating is improved remarkably by the simple chemical treatment, since the surface of the alkali-treated MAO coating could provide abundant Ti-OH groups probably formed by ionic exchanges between (Ca2+, Na+) ions of the alkali-treated MAO coating and H3O+ ions of a simulated body fluid (SBF). Moreover, Ca released from the alkali-treated MAO coating increases the degree of supersaturation of SBF, promoting the formation of apatite. The apatite induced by the alkali-treated MAO coating possesses carbonated structure and pore networks on the nanometer scale.     

Role of glass structure in defining the chemical dissolution behavior,bioactivity and antioxidant properties of zinc and strontium co-doped alkali-free phosphosilicate glasses

We investigated the structure–property relationships in a series of alkali-free phosphosilicate glass compositions co-doped with Zn²⁺ and Sr²⁺. The emphasis was laid on understanding the structural role of Sr²⁺ and Zn²⁺ co-doping on the chemical dissolution behavior of glasses and its impact on their in vitro bioactivity. The structure of glasses was studied using molecular dynamics simulations in combination with solid state nuclear magnetic resonance spectroscopy. The relevant structural properties are then linked to the observed degradation behavior, in vitro bioactivity, osteoblast proliferation and oxidative stress levels. The apatite-forming ability of glasses has been investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy–energy-dispersive spectroscopy after immersion of glass powders/bulk in simulated body fluid (SBF) for time durations varying between 1 h and 14 days, while their chemical degradation has been studied in Tris–HCl in accordance with ISO 10993-14. All the glasses exhibit hydroxyapatite formation on their surface within 1–3 h of their immersion in SBF. The cellular responses were observed in vitro on bulk glass samples using human osteosarcoma MG63 cell line. The dose-dependent cytoprotective effect of glasses with respect to the concentration of zinc and strontium released from the glasses is also discussed.     

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.     

A comparative study of in vitro apatite deposition on heat-, H(2)O(2)-, and NaOH-treated titanium surfaces

Commercially pure titanium specimens are subjected to three different treatments, and their bioactivity are evaluated by immersing the specimens in a simulated body fluid (SBF, Kokubo's recipe) for various periods up to 7 days, with particular attention being paid to the differences in apatite deposition between surfaces open to SBF and surfaces in contact with the container's bottom. The treatment with a H(2)O(2)/HCl solution at 80 degrees C for 30 min followed by heating at 400 degrees C for 1 h produces an anatase titania gel layer on the specimen surface. This gel layer deposits apatite both on the contact and on open surfaces, and apatite deposition ability does not change with pre-staking in distilled water. The treatment with a NaOH solution at 60 degrees C for 3 days produces a sodium titanate gel layer. This gel layer can deposit apatite only on the contact surface, and the apatite deposition ability is completely lost after 1 day of pre-staking in distilled water. It is concluded, therefore, that the bioactivity of the titania gel originates from the favorable structure of the gel itself while the bioactivity of the sodium titanate gel depends heavily on ion release from the gel. The third treatment, a simple heat treatment at 400 degrees C for 1 h, produces a dense (not porous) oxide layer on the specimen surface. The specimens can deposit apatite on the contact surface after only 3 days of staking in SBF, but they cannot deposit apatite on the open surface for up to 2 months of staking. The implications of such apatite deposition behavior have been discussed in relation to the environments of titanium implants in bone as well as to the methodology of the SBF staking experiment.     

Apatite formation on zirconium metal treated with aqueous NaOH.

Previous studies by the authors have shown that titanium metal, titanium alloys and tantalum metal which were subjected to aqueous NaOH solution and subsequent heat treatments form an apatite surface layer upon immersion in a simulated body fluid (SBF) with ion concentrations nearly equal to those in human blood plasma. These metals form the apatite surface layer even in living body, and bond to living bone through the apatite layer. In the present study, the apatite-forming ability of NaOH-treated zirconium metal in SBF has been investigated. A hydrated zirconia gel layer was formed on the surface of the zirconium metal on exposure to 1-15 M NaOH aqueous solutions at 95 degrees C for 24h. It was observed that the metals treated in NaOH aqueous solutions with concentrations above 5 M form an apatite layer on their surface in SBF. This indicates that the Zr-OH group of the zirconia gel induces apatite nucleation. The present study points to the possibility of obtaining bioactive zirconium after treatment by NaOH.     

Apatite-forming ability and mechanical properties of PTMO-modified CaO-SiO2 hybrids prepared by sol-gel processing: effect of CaO and PTMO contents

Transparent monolithics of triethoxysilane end-capped poly(tetramethylene oxide) (Si-PTMO)-modified CaO-SiO2 hybrids were successfully synthesized by hydrolysis and polycondensation of Si-PTMO, tetraethoxysilane (TEOS) and calcium nitrate. As for the samples with varying (Ca(NO3)2)/(TEOS) molar ratios under constant ratio of (Si-PTMO)/(TEOS) of 2/3 in weight. the apatite-forming ability in a simulated body fluid (SBF) which is indicative of bioactivity. remarkably increased with increasing CaO content, although the tensile strength and Young's modulus decreased. The hybrid with (Ca(NO3)2)/(TEOS) = 0.15 in mol formed an apatite on its surface within only 1 day. For this series of samples, the strain at failure which is a measure of capability for deformation of material, was found to be about 30% and almost independent of CaO content. As for the samples with varying (Si-PTMO)/(TEOS) weight ratios under constant ratio of (Ca(NO3)2)/(TEOS) of 0.15 in mol, the strain at failure increased with increasing Si-PTMO content, but the apatite-forming ability, tensile strength and Young's modulus decreased. Thus, the synthesis of the hybrids exhibiting both high apatite-forming ability and high extensibility can be achieved by selecting suitable CaO and Si-PTMO contents. These new kind of hybrid materials may be useful as bioactive bone-repairing materials.     

Biological evaluation of an apatite-mullite glass-ceramic produced via selective laser sintering

The biological performance of a porous apatite-mullite glass-ceramic, manufactured via a selective laser sintering (SLS) method, was evaluated to determine its potential as a bone replacement material. Direct contact and extract assays were used to assess the cytotoxicity of the material. A pilot animal study, implanting the material into rabbit tibiae for 4 weeks, was also carried out to assess in vivo bioactivity. The material produced by SLS did not show any acute cytotoxic effects by either contact or extract methods. There was no evidence of an apatite layer forming on the surface of the material when soaked in SBF for 30 days, suggesting that the material was unlikely to exhibit bioactive behaviour in vivo. It is hypothesized that the material was unable to form an apatite layer in SBF due to the fact that this glass-ceramic was highly crystalline and the fluorapatite crystal phase was relatively stable in SBF, as were the two aluminosilicate crystal phases. There was thus no release of calcium and phosphorus and no formation of silanol groups to trigger apatite deposition from solution within the test time period. Following implantation in rabbit tibiae for 4 weeks, bone was seen to have grown into the porous structure of the laser-sintered parts, and appeared to be very close to, or directly contacting, the material surface. This result may reflect the local environment in vivo compared to that artificially found with the in vitro SBF test and, furthermore, confirms previous in vivo data on these glass-ceramics.     

In vitro bioactivity evaluation of titanium and niobium metals with different surface morphologies

Current orthopaedic biomaterials research mainly focuses on designing implants that could induce controlled, guided and rapid healing. In the present study, the surface morphologies of titanium (Ti) and niobium (Nb) metals were tailored to form nanoporous, nanoplate and nanofibre-like structures through adjustment of the temperature in the alkali-heat treatment. The in vitro bioactivity of these structures was then evaluated by soaking the treated samples in simulated body fluid (SBF). It was found that the morphology of the modified surface significantly influenced the apatite-inducing ability. The Ti surface with a nanofibre-like structure showed better apatite-inducing ability than the nanoporous or nanoplate surface structures. A thick dense apatite layer formed on the Ti surface with nanofibre-like structure after 1 week of soaking in SBF. It is expected that the nanofibre-like surface could achieve good apatite formation in vivo and subsequently enhance osteoblast cell adhesion and bone formation.     

Preparation of bioactive titanium metal via anodic oxidation treatment

Titania with specific structures of anatase and rutile was found to induce apatite formation in vitro. In this study, anodic oxidation in H(2)SO(4) solution, which could form anatase and rutile on titanium metal surface by conditioning the process, was employed to modify the structure and bioactivity of biomedical titanium. After the titanium metal was subjected to anodic oxidation treatment, thin film X-ray diffraction and scanning electron microscopy results showed the titanium metals surfaces were covered by porous titania of anatase and/or rutile. In simulated body fluid (SBF), the titanium anodically oxidized under the conditions with spark-discharge could induce apatite formation on its surface. The induction period of apatite formation was decreased with increasing amount of either anatase or rutile by conditioning the anodic oxidation. After the titanium metal, anodically oxidized under the conditions without spark-discharge, was subjected to heat treatment at 600 degrees C for 1 h, it could also induce apatite formation in SBF because the amount of anatase and/or rutile was increased by the heat treatment. Our results showed that induction of apatite-forming ability on titanium metal could be attained by anodic oxidation conjoined with heat treatment. So it was believed that anodic oxidation in H(2)SO(4) solution was an effective way to prepare bioactive titanium.     

In vitro degradation, bioactivity, and cytocompatibility of calcium silicate, dimagnesium silicate, and tricalcium phosphate bioceramics

CaSiO3 (CS) ceramics have been regarded as a potential bioactive material for bone regeneration. Mg2SiO4 (M2S) ceramic has been reported as a novel bioceramic with higher mechanical properties and good biocompatibility recently. beta-Ca2(PO4)2 (beta-TCP) ceramic is a well-known bioactive and degradable material for bone repair. The aim of this study is to investigate and compare the effect of three bioceramics with different chemical composition on the in vitro degradation, apatite-forming ability in simulated body fluid (SBF) and cytocompatibility. The degradation was evaluated through the activation energy of Si or P ion released from ceramics and the weight loss of the ceramics in Tris-HCl buffer solution. Formation of bone-like apatite on different bioceramic surfaces was investigated in SBF. The presence of bone-like apatite layer on the material surface after soaking in SBF was demonstrated by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM). The effect of ionic products from the three kinds of material dissolution on osteoblast-like cell proliferation was investigated. The results showed that the degradation rate of CS was much faster than that of beta-TCP and M2S ceramics. Apatite formation occurred on the CS ceramics quickly. However, it was less likely to occur on the surfaces of beta-TCP and M2S ceramics. The ionic products from extracts of CS and M2S could stimulate osteoblast-like cell proliferation at certain concentration range throughout the 6-day culture period.     

Preparation of SBF with different HCO3- content and its influence on the composition of biomimetic apatites

The bioactivity of bone and dental implant materials is usually tested in vitro using simulated body fluid (SBF). The composition of common SBF differs from that of blood plasma in that it has a higher Cl- and a lower HCO3- concentration, which affects the composition of in vitro formed bone-like apatite. Five different SBFs with a composition of 142 Na+, 5 K+, 2.5 Ca2+, 1 Mg2+, 1SO4(2-), 1HPO4(2-), and 136 (Cl-+HCO3-) mmol/l were prepared with HCO3- concentrations ranging from 5 to 27 mmol/l. The SBF solutions were prepared by mixing stable concentrated solutions, which increase the reproducibility of in vitro tests due to negligible changes of pH during preparation. The high stability of thus prepared SBF enables the evaluation of hydroxyapatite formation on the surface of bioactive materials without the negative effect of spontaneous precipitation. Furthermore, the use of concentrated solutions offers a facile way to prepare SBF with different ionic contents and thus modify the composition of Ca-P layers precipitated on the surface of the bioactive materials exposed to the SBF solutions. The SBF solutions were shown to be supersaturated with respect to slightly carbonated apatite. The Fourier transform infrared (FT-IR), Raman and X-ray analyses of the precipitated layers indicate that the HCO3- content in SBF influences the composition and structure of the calcium phosphates obtained. It can be supposed that as long as the HCO3- concentration in the testing solutions is lower than 20 mmol/l, only B-type HCA precipitates. At higher HCO3- concentrations, it can be assumed that A-type HCA forms as well considering FT-IR, Raman and X-ray measurements.     

Bone-bonding ability of P2O5-free CaO.SiO2 glasses

An apatite- and wollastonite-containing glass-ceramic (A.W-GC) has been reported to form a tight bond with living bone through an apatite layer formed on its surface. This layer is considered to be formed by dissolution of Ca2+ and HSiO3- ions from the glass-ceramic into the surrounding body fluids. In order to confirm this proposed mechanism for the surface reaction of A.W-GC, three kinds of glass in the systems CaO-SiO2, CaO-SiO2-CaF2, and CaO-SiO2-P2O5 were implanted into the tibiae of rabbits for 3 or 8 weeks. Contact microradiography and SEM-EPMA showed that all three kinds of glass formed a Ca,P-rich layer in combination with a Si-rich layer on their surfaces within 3 weeks and formed a direct bond with bone via these layers. The detaching test, performed 8 weeks after implantation, showed that the loads required to detach the implants from the bone were almost equal for the phosphorus-free and the phosphorus-containing glasses. It was concluded that even P2O5-free CaO.SiO2 glass formed a Ca,P-rich layer on its surface and bonded tightly with living bone. If glasses and glass-ceramics release at least Ca2+ and HSiO3- ions, this would be sufficient for them to form the Ca,P-rich layer on their surfaces in vivo, enabling them to bond directly with bone.