evaluation of the In Vitro Bioactivity of Bioceramics

Two common methods have been used to evaluate the in vitro bioactivity of bioceramics for the application of bone repair. One is to evaluate the ability of apatite formation by soaking ceramics in simulated body fluids (SBF); the other method is to evaluate the effect of ceramics on osteogenic differentiation using cell experiments. Both methods have their own drawbacks in evaluating the in vitro bioactivity of bioceramics. In this commentary paper we review the application of both methods in bioactivity of bioceramics and conclude that (i) SBF method is an efficient method to investigate the in vitro bioactivity of silicate-based bioceramics, (ii) cellular bioactivity of bioceramics should be investigated by evaluating their stimulatory ability using standard bioceramics as controls; and (iii) the combination of these two methods to evaluate the in vitro bioactivity of bioceramics can improve the screening efficiency for the selection of bioactive ceramics for bone regeneration.

Bone and Tissue Regeneration Insights 2009:2

Introduction
The bioactivity of ceramics has been defined as "the bond ability with host bone tissue". 1 This includes enhancing the ability of apatite formation, osteoblast differentiation and bone matrix formation.Current bioceramics, such as hydroxyapatite, β-tricalcium phosphate (β-TCP), HAp/β-TCP and bioglass 45S5 ® have been widely used as bone repair materials, mainly due to their excellent bioactivity which makes them capable of bonding closely with the host bone tissue.The main disadvantage of these bioceramics is their relatively low mechanical strength, particularly low fracture toughness, which limits their application to only low-load bearing areas in the human body.To develop new bioactive ceramics for load bearing bone repair applications, it is important to understand the bonding of ceramics to living bone and methods to test bonding abilities. 2In order to avoid the high cost of in vivo experiment, several in vitro tests have been used to predict the in vivo bone bioactivity of bioceramics.However, there is still challenging to evaluate the in vitro bioactivity of bioceramics.Currently, two common methods have been used for testing the in vitro bioactivity of bioceramics.6][7] To evaluate apatite formation, Kokubo and colleagues have established a method to examine the apatite formation on materials in SBF.This method is useful prior to doing in vivo bone bioactivity experiments and can significantly reduce the number of animals needed for in vivo evaluation. 2 However, Bohner and colleagues recently published a study which showed that there is currently not enough scientific evidence to support Kokubo's claims that SBF is a useful tool to evaluate the in vitro bioactivity, and that the choice of SBF solution for testing the in vitro bioactivity of bioceramics is quite arbitrary. 8As for the cell experiment method, a large part of the scientific community has accepted the paradigm that in vitro cell testing can be used to test the in vitro bioactivity of bioceramics.This method has been widely used in testing the bioactivity of bioceramics.][11][12] The methods for evaluating the in vitro bioactivity of bioceramics are still not clear.Therefore, the aim of this commentary paper is to present our view of how best to evaluate in vitro bioactivity of bioceramics.

comments for the In Vitro Bioactivity of Bioceramics
Firstly, the SBF method is a useful way to test the in vitro bioactivity of bioceramics for the assessment of the apatite formation potential.However, the reliability this method depends on the category of bioceramics tested.Silicate-based bioceramics, including silicate bioglass 45S5 @ , 1 wollastonite (CaSiO 3 ), 4,13 akermanite (Ca 2 MgSi 2 O 7 ) (See Fig. 1) 3 and diopside (Ca 2 MgSi 2 O 6 ) 14 ceramics, have been shown to have excellent apatite forming abilities in SBF.Other studies also showed that these silicate ceramics possess good in vivo bioactivity, [15][16][17][18] which indicates that SBF testing is an efficient method to evaluate their in vitro bioactivity.Phosphate-based bioceramics (HAp and β-TCP), [19][20][21] carbonate-based bioceramics (coral, CaCO 3 ), and sulfate-based materials (CaSO 4 ), have no obvious apatite formation when soaked in SBF for a short time.They do, however, have excellent in vivo bone formation abilities, 22,23 but this suggests that SBF alone is not sufficient to test the in vitro bioactivity for these three bioceramics.The SBF method, therefore, is useful for evaluating the in vitro bioactivity of silicate ceramics, but not for other types of bioceramics.The possible reason for this is that the biochemistry of in vivo bone formation of these bioceramics is significantly different.Silicate-based bioceramics bond with host bone via the formation of bone-like apatite layers due to the dissolution Ca 2+ or other metal ions, followed by the deposition of Ca-P in the body. 1,17,24,25The phenomenon of dissolution and deposition does in fact happen in the SBF solution for silicate-based bioceramics.Sintered HAp and β-TCP ceramics can also bond directly with host bone. 15,21,26heir apatite-formation ability mainly depends on their crystanillity and sintering property.Fully sintered HAp bulk ceramics are difficult to induce bone-apatite formation, 19,20 and sintered β-TCP ceramics exhibit a poor ability of inducing apatite formation; 27 however, HAp particles can induce apatite formation. 28CaCO 3 and CaSO 4 materials bond to living bone, which may be related to their high resorbability. 2 Secondly, cell experiments have been used widely to investigate the in vitro bioactivity of bioceramics.It is known that Al 2 O 3 , ZrO 2 , TiO 2 , and Mg 2 SiO 4 bioceramics have been considered as bioinert ceramics since they cannot induce apatite formation in SBF.12]29,30 There are therefore a number of ceramics which elicits excellent cell responses; however, this does not necessarily translate into good in vivo bioactivity.On the contrary, some ceramics, such as highly degradable CaSiO 3 ceramics, are detrimental to the in vitro growth of human osteoblasts due to their high rate of dissolution which results in a high localized pH environment. 7,31,32On the other hand, recent studies have shown that CaSiO 3 ceramics possess excellent in vivo bone-formation ability and their in vivo bioactivity is greater than that of β-TCP. 17Cell based experiments to evaluate in vitro bioactivity of bioceramics are therefore not completely reliable.In addition, if cell cultures are used to evaluate the in vitro bioactivity of bioceramics, one should also investigate if the same bioceramics have the ability to stimulate or enhance a cell response.The selection of positive control materials to compare the cellular response is therefore important.Previous studies have selected β-TCP ceramics as the control material to compare the osteoblast response to akermanite (Ca 2 MgSi 2 O 7 ) bioceramics. 6,33It is necessary to know that the standard β-TCP ceramics has been carefully prepared by a standardized method and procedure, since the β-TCP ceramics can be prepared by a number of methods and these will affect different cell responses.Other studies have used blank tissue culture plate as control and to show that the ionic products of bioglass, 5 akermanite, 3 Sr-CaSiO 3 13 and hardystonite (Ca 2 ZnSi 2 O 7 ) 34 ceramics stimulate osteoblast proliferation.Tissue culture plate can therefore be regarded as one of the standard controls to evaluate in vitro bioactivity of bioceramics.
Thirdly, combining SBF and cell experiments to evaluate the in vitro bioactivity of bioceramics may be the better option.This is because if a novel bioceramic not only has the ability to induce apatite deposition in SBF, but also stimulates a cell response, such a bioceramic would most likely possess excellent in vivo bioactivity as well.Our own work has shown that akermanite ceramics has excellent apatite-forming abilities in SBF 3 and significantly enhances in vitro osteoblast attachment (See Fig. 2), proliferation, differentiation and gene express compared to β-TCP ceramics, 6 and in vivo experiments have confirmed that they also have excellent bone-forming abilities in animal tests. 167][38] The in vivo experiment has also shown that 45S5 bioglass has JeoL SeI 20.0kV 10µm WD 1mm ×1,000 excellent bone bond ability to be used as a bone repair material. 1,15,39nclusions Selection of methods for the evaluation of in vitro bioactivity of bioceramics depends on the composition of bioceramics and the mechanism of their bone formation.The synthetic body fluid method is a useful approach to evaluate the in vitro bioactivity of silicate-based bioceramics.Cell based experiments is also a valuable test for bioactivity of bioceramics, but relevant standard materials should be considered as positive control.We recommend the combination of SBF and cell testing methods to evaluate the in vitro bioactivity of bioceramics, an approach which will improve the efficiency of screening bioceramics for further in vivo evaluation of bone repair.

Figure 2 .
Figure 2. osteoblast-like cells growing on the surface of akermnaite ceramics after 7 days of culture.