7
Simulated Body Fluid (SBF) as a Standard Tool to Test the Bioactivity of Implants
Tadashi Kokubo and Hiroaki Takadama
Abstract
Most bone-bonding bioactive materials form bone-like apatite on their surfaces after being implanted into the living body, and bond to neighboring bone through this apatite layer. The apatite layer can be reproduced on the surfaces of materials in an organic-substance-free acellular simulated body fluid (SBF) with ion con- centrations almost equal to those of human blood plasma. The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on the material in SBF. In this chapter, the validity of this method for evaluating the bone-bonding bioactivity of a material, the ion concentrations of SBF, the materials able to form apatite, the characteristics of apatite, the bone-bonding mechanisms of bioactive materials, and the mechanisms of apatite formation, are reviewed.
Key words: simulated body fluid (SBF), bioactive material, apatite-forming abil- ity, bone-bonding ability, bone substitute, bone-like apatite.
7.1
Introduction
Various materials, including Bioglass [1], sintered hydroxyapatite [2], sintered beta-tricalcium phosphate [3], biphasic ceramics of hydroxyapatite and trical- cium phosphate [4], and glass–ceramic A-W [A¼apatite (Ca10(PO4)6(O, F2));
W¼wollastonite (CaOSiO2)] [5], can bond to living bone. These are referred to as ‘‘bioactive’’ materials, and many are currently in clinical use as important bone substitutes. Most of them bond to living bone through an apatite layer that forms on their surfaces after implantation into the living body. This apatite formation has been reproduced on their surfaces in an organic-substance-free acellular simulated body fluid (SBF), with ion concentrations almost equal to those of human blood plasma [6]. This indicates that the bone-bonding bioactivity of a
97
Handbook of Biomineralization. Edited by M. Epple and E. Ba¨uerlein Copyright82007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31806-3
material can be evaluated in preliminary fashion, before conducting animal ex- periments, by examining apatite formation on its surface in SBF. As a result, the number of animals required to evaluate the bone-bonding ability of a material can be reduced, and today many laboratories utilize SBF as a standard tool when testing the bioactivity of new materials. In this chapter, the correlation between the bone-bonding bioactivity of materials and their apatite-forming ability in SBF, the ion concentrations of SBF, the dependence of apatite formation on the material, the characteristics of apatite, the bone-bonding mechanisms of bio- active materials, and the mechanisms of apatite formation on these materials, are described.
7.2
Qualitative Correlation of Bone-Bonding Bioactivity of a Material with Apatite Formation on its Surface in SBF
Materials that bond to living bone through an apatite or calcium phosphate layer that forms on their surfaces after implantation into the living body include:
a Bioglass 45S5 type-glass in a Na2OaCaOaSiO2aP2O5
system [7];
bioactive glasses in the Na2OaCaOaB2O3aAl2O3aP2O5 system [8];
glasses in the CaOaSiO2system [9];
Ceravital-type glass–ceramics containing crystalline apatite in the Na2OaCaOaSiO2aP2O5system [10];
Glass–ceramic A-W, containing crystalline apatite and wollastonite, in the MgOaCaOaSiO2aP2O5system [11];
Bioverite-type glass–ceramics containing crystalline apatite and fluorophlogopite in the
Na2OaMgOaCaOaAl2O3aSiO2aP2O5aF system [12];
sintered hydroxyapatite [13];
biphasic ceramics of hydroxyapatite and beta-tricalcium phosphate;
sintered calcium sulfate [14];
a composite of glass–ceramic A-W with polyethylene [15];
titanium metal subjected to NaOH and heat treatments [16];
and
tantalum metal subjected to NaOH and heat treatments [17].
An example of an interface of glass–ceramic A-W to living bone is shown in Fig- ure 7.1.
All of these bone-bonding bioactive glasses, glass–ceramics, sintered crystal- line ceramics, composites and metals have been confirmed as forming an apa- tite on their surfaces in SBF within 4 weeks [4, 6–8, 10, 14, 18–23], except for the Bioverite-type glass–ceramic, which has not been investigated for apatite forma-
tion on its surface in SBF. An apatite layer formed on a glass–ceramic A-W in SBF is illustrated in Figure 7.2.
When a small amount of Al2O3was added to the composition of Bioglass-type glass [24], CaOaSiO2glass [25], and glass–ceramic A-W [26], the resultant glasses and glass–ceramics did not form an apatite or calcium phosphate layer on their surfaces in the living body, and did not bond to the neighboring bone. In addi- tion, none of these materials with added Al2O3formed apatite on their surfaces within 4 weeks in SBF [14, 27, 28].
It can be concluded from these results that the essential requirement for a material to bond to living bone is the formation of an apatite or calcium phos- phate layer on its surface, and that the bone-bonding bioactivity of a material can be evaluated by examining the formation of an apatite layer on its surface in SBF.
However, it should be noted here that a small number of cases in which a ma- terial bonds to living bone without yielding a detectable apatite layer at their inter- faces have been reported. Sintered beta-tricalcium phosphate and a natural calcite of calcium carbonate are examples [29, 30], with neither material forming an apa- tite layer on its surface within 4 weeks in SBF [31, 32]. In fact, the bone-bonding properties of these materials might be related to their high resorbability in the living body.
One case in which a material – abalone shell – does not bond to living bone, despite forming an apatite or calcium phosphate layer on its surface in the living Fig. 7.1Transmission electron microscopy image of the interface of
glass–ceramic A-W and rabbit tibial bone [13].
7.2 Qualitative Correlation of Bone-Bonding Bioactivity of a Material with Apatite Formation 99
body – has been also reported. Abalone shell also forms an apatite layer on its sur- face in SBF [32], and suppression of the bone-bonding bioactivity of this material might be attributed to foreign body reactions elicited by small amounts of protein contained in the shell.
From the above findings, it can be concluded that a material which is able to form an apatite layer on its surface in SBF may bond to living bone through the apatite layer that forms on its surface, as long as the material does not release any component that induces toxic or immune responses in the surrounding tissue.
Based on these findings, the examination of apatite formation on a surface of a material in SBF would be a useful tool for predicting the bone-bonding bioactivity of a material, before progressing to animal experiments. Indeed, by using this method not only the number of animals but also the duration of animal experi- ments required to evaluate the bone-bonding bioactivity of a material can be greatly reduced.
7.3
Quantitative Correlation of Bone-Bonding Bioactivity and Apatite-Forming Ability in SBF
Not all bioactive materials show equal bone-bonding ability; rather, the time re- quired for a material to bond to living bone, and the amount of bone formed Fig. 7.2 The apatite layer formed on glass–ceramic A-W in simulated body fluid [19].
around a material in a given time, will vary widely depending on the material in- volved. The time required for a bioactive material to become fully covered with apatite in SBF also varies, depending on the material. In order to investigate the relationship between bone formationin vivoand apatite formation in SBF, bone formation in defects in rabbit femurs filled with Na2OaCaOaSiO2glass particles (the SiO2 contents of which were changed from 70.0 to 50.0 mol%, with a constant Na2O/CaO molar ratio of one) were examined [33]. The time required for the same glasses in SBF to form bone-like apatite which fully covered their surfaces was also measured [34]. The data provided in Figure 7.3 show clearly that bone formation around glass particles increases with the increasing apatite- forming ability of the glasses in SBF. This, in turn, indicates that the bone- bonding bioactivity of a material can be evaluated not only qualitatively but also quantitatively, by examining the apatite-forming ability on the material’s surface in SBF.
7.4
Ion Concentrations of SBF
In all of the above-described investigations, the organic-substance-free acellular solution used as the SBF had ion concentrations as first reported by Kokubo et al. in 1990 [6], and as corrected by the same authors in 1991 [35]. However, the ion concentrations of this SBF were not exactly equal to those of human blood plasma (see Table 7.1), as SBF is richer in Cl ions and poorer in HCO3 ions than is human blood plasma [36]. In 2003, Oyane et al. proposed a revised simu- lated body fluid (r-SBF), in which the ion concentrations were identical to those of human blood plasma [37]. However, r-SBF had a strong tendency to produce Fig. 7.3The rate of bone formation on a cross-section of a defect of
rabbit femur when filled with glass particles 6 weeks after implantation compared with time of surface apatite formation in simulated body fluid [33].
7.4 Ion Concentrations of SBF 101
precipitates of calcium carbonate, as it is highly supersaturated with respect to hydroxyapatite and calcite [38].
In 2004, the method for preparing conventional SBF was further refined and simplified such that it could be easily prepared and subjected to round-robin test- ing by 10 research institutes [39]. This refined SBF recipe (the details of which have been published [40]) was proposed to the International Organization for Standardization as a standard solution for in-vitro monitoring of the apatite- forming ability of implant materials.
Simulated body fluids with higher ion concentrations (e.g., 1.5 and 4 SBF, where ion concentrations are 1.5- or four-times those of SBF) have also been used to evaluate the bone-bonding abilities of materials, or the production of a bone-like apatite layer on materials. It should be noted, however, that no corre- lation has been identified between apatite formation in such solutions and bone-bonding ability, and that the apatite formed in these solutions differs in composition from bone mineral [41].
7.5
Materials Able to Form Apatite
Despite human body fluid being highly supersaturated with respect to apatite (even under normal conditions [42]), apatite does not usually precipitate in the living body, except at sites of bone tissue, as the energy barrier for its nucleation is high. This means that, once apatite nuclei have been formed catalytically on a material, they can grow spontaneously by consuming the calcium and phosphate ions from the surrounding body fluid.
The question persists, however, as to what type of material induces apatite nucleation. In an attempt to answer this question, various types of gels prepared using sol-gel methods were soaked in SBF, and their apatite-forming abilities ex- amined. Although SiO2[43], TiO2[44], ZrO2[45], Nb2O5[46] and Ta2O5[47] gels were seen to form apatite on their surfaces, Al2O3 [44] gels did not (Fig. 7.4), which indicated that the SiaOH, TiaOH, ZraOH, NbaOH and TaaOH groups that were abundant on the surfaces of the gels were effective in inducing apatite Table 7.1 Ion concentrations of simulated body fluid (SBF) and human blood plasma.
Ion concentration [mmol]
NaB KB Mg2B Ca2B ClC HCO3C HPO42C SO42C
Blood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
SBF 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5
nucleation. Subsequently, Tanahashi et al., using self-assembled monolayers, showed that COOH and PO4H2groups were also effective for apatite nucleation [48].
Based on these findings, titanium metal and its alloys were subjected to NaOH solution and heat treatment to form sodium titanate on their surfaces. These treated materials were found to form bone-like apatite on their surfaces in the living body, and to bond to living bone [49], and subsequently were applied for use in hip-joint prostheses.
7.6
Composition and Structure of Apatite
The calcium phosphate layer formed on bioactive materials after implantation into living bodies has been identified by micro X-ray diffraction [50] and electron [51] diffraction as a nanosized crystalline apatite. However, it has been difficult to obtain more detailed structural information for these calcium phosphate layers formedin vivo.
More detailed structural information can be obtained for apatite formed on bioactive materials in SBF. According to observations made with transmission electron microscopy (TEM), the apatite on both glass–ceramic A-W [52] and NaOH- and heat-treated titanium metal [53] in SBF takes the shape of thin nee- dles of 10 nm thickness and 100 nm length (Fig. 7.5). This apatite has a Ca/P atomic ratio of about 1.65, which is less than the stoichiometric value of 1.67, and contains a small amount of Naþ and Mg2þ ions beside CO32 ions [19, 52, 53]. As these characteristics are similar to those of bone mineral, the material may be referred to as ‘‘bone-like’’ apatite.
Fig. 7.4Apatite formed on (left) SiO2and (right) TiO2gels in simulated body fluid [44].
7.6 Composition and Structure of Apatite 103
7.7
Mechanism of Bonding of Bioactive Material to Bone
As described above, most bioactive materials form apatite on their surfaces after being implanted into the living body. As this surface apatite is very similar to bone mineral in its composition, structure and morphology, the bone-producing cells (osteoblasts) could preferentially proliferate and differentiate on its surface to produce collagen and apatite, similar to their behavior on the surface of frac- tured bone (Fig. 7.6) [54]. As a result, the surrounded bone may come into direct Fig. 7.5 Transmission electron microscopy (left) and energy dispersive
X-ray analysis (right) images of apatite formed on NaOH- and heat- treated titanium metal in simulated body fluid [53].
Fig. 7.6 Transmission electron microscopy image of the interface of glass–ceramic A-W and rabbit tibial bone at early stage after implantation [54].
contact with the surface apatite layer on materials. When this occurs, a tight chemical bond is formed between the apatite in the bone and the surface apatite to reduce their interface energy.
7.8
Mechanisms of Apatite Formation
If apatite formation on bioactive materials implanted into the living body can be reproduced on their surfaces in SBF, then the mechanisms of apatite formation on the materials might be revealed by the examining surface structural changes of the materials as a function of soaking time in SBF.
Based on TEM observations and zeta potential measurements, the mechanism of apatite formation on sintered hydroxyapatite in body environment is inter- preted as follows [55]. The sintered hydroxyapatite is initially negatively charged on its surface, and combines with positively charged Ca2þ ions in the surround- ing fluid. As a result, Ca-rich amorphous calcium phosphate is formed on the sintered hydroxyapatite. As the Ca2þ ions accumulate, the sintered hydroxyapa- tite becomes positively charged on its surface and reacts with negatively charged phosphate ions. As a result, Ca-poor amorphous calcium phosphate is formed which is eventually transformed into the more stable, nanosized crystalline bone-like apatite. This mechanism is essentially the same in fluids containing proteins [56].
Apatite formation on NaOH- and heat-treated titanium metal in a body envi- ronment is similarly interpreted as follows [53, 57]. The treated titanium metal releases Naþions from its surface sodium titanate layer via exchange with H3Oþ ions in the fluid, to form TiaOH groups (Fig. 7.7). As a result, its surface becomes negatively charged and reacts with positively charged Ca2þ ions to form calcium titanate. As the calcium ions accumulate, the positively charged surface reacts with negatively charged phosphate ions, forming amorphous cal- cium phosphate. As this phase is metastable, it eventually transforms into nano- sized, crystalline bone-like apatite.
Fig. 7.7The mechanism of bone-like apatite formation on NaOH- and heat-treated titanium metalin vitro[57]. SBF¼simulated body fluid.
7.8 Mechanisms of Apatite Formation 105
7.9 Summary
Simulated body fluid, which is used to test the bone-bonding bioactivity of various materials, is identical to human blood plasma in terms of its ion concentrations, but does not contain organic substances such as proteins. Nevertheless, the soak- ing of bioactive materials in SBF can reproduce the apatite formation seen on such materials in the living body. SBF is easily prepared and relatively stable at body temperature. Moreover, it is useful for evaluating the bone-bonding bioactiv- ity of new materials and investigating the mechanisms of apatite formation on, and bone bonding of, materials.
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