Leading OpinionCan bioactivity be tested in vitro with SBF solution?☆,☆☆
Introduction
Two decades ago, Kokubo et al. [1], [2] used SBF to perform in vitro simulations of in vivo conditions. In 2003 a revised SBF solution was proposed to take into account the fact that a large proportion of calcium and magnesium species present in serum is bound to proteins and hence unavailable for apatite precipitation [3]. The revised SBF solution had a 40% lower calcium concentration and a 33% lower magnesium concentration.
Since 1987, the use of SBF for bioactivity testing has exploded. A search in Scopus (www.scopus.com) using the keywords “bioactivity” and “simulated body fluid” (in all fields) leads to 1975 hits (Jan 5, 2009) with 379 hits in 2008. In 2006, Kokubo and Takadama reviewed the topic in a paper entitled “How useful is SBF in predicting in vivo bone bioactivity?” [1] and reiterated the statement that SBF could be used to test bioactivity. After 20 years of research in this field, the opinion shared by a large part of the biomaterials community is that the formation of apatite on a material dipped in SBF is a proof of its bioactivity and can be used to anticipate its bone bonding ability in vivo.
According to the ESB consensus conference of 1987 [4], a bioactive material is “one which has been designed to induce specific biological activity”. Obviously, there has been a drift of meaning over time, because Kokubo and Takadama [1] consider bioactive materials as bone bonding materials. More specifically, these authors state that “…the essential requirement for a material to bond to living bone is the formation of bone-like apatite on its surface when implanted in the living body”, and that “…this in vivo apatite formation can be reproduced in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma.” Thus, according to Kokubo and Takadama's definition of bioactivity, a bioactive material is a material on which bone-like hydroxyapatite will form selectively after it is immersed in a serum-like solution [1], [5]. Despite the absence of recommendation of Kokubo regarding the CO2 partial pressure (p(CO2)), it is worth mentioning that the use of physiological conditions implies that the test should be performed at p(CO2) = 0.05 atm (5%) since human serum is in equilibrium with such a partial pressure.
Section snippets
Crystallization theory
Crystallization theory suggests that bone bonding ability (or bioactivity) and apatite formation in SBF solution are two distinct phenomena. To explain this statement, it is necessary to review basic principles of crystallization theory and relate these principles to the SBF-based bioactivity test.
Thermochemical calculations show that serum and SBF are supersaturated towards apatite crystals [6]. In other words, the system is metastable and will eventually become thermodynamically stable by
Apatite formation in SBF and crystallization theories
Relating these theories to dipping tests in SBF, it becomes clear that a “bioactive” compound according to Kokubo's definition [1], [5] is a material that accelerates heterogeneous apatite crystallization in a solution supersaturated towards hydroxyapatite. This can be achieved by several strategies: (i) providing apatite nuclei that remove the need to nucleate apatite crystals, (ii) providing a surface with a low interfacial energy with apatite, or (iii) changing the local supersaturation
Bioactivity testing using SBF
In their “leading opinion paper”, Kokubo and Takadama [1] concluded that “a material able to have apatite form on its surface in SBF has apatite produced on its surface in the living body, and bonds to living bone through this apatite layer”. This conclusion is contradicted by the observation done with CSH and DCPD, which both show an apatite layer forming in SBF [10], [24] but no direct bone bonding in vivo [21], [22], [23], [25]. These authors also concluded that “examination of apatite
Conclusion
In this paper, it has been shown that the choice of SBF solution for testing the bone bonding ability of materials is arbitrary. Moreover, the protocol proposed by Kokubo and Takadama [1] for preparing SBF solutions leaves room for improvement, since (i) the procedure is long and tricky, (ii) the solutions are not filtered, and (iii) the carbonate content is not controlled. Furthermore, the use of SBF for bioactivity testing leads to false positive and false negative results. Despite these
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Editor's Note: This paper is one of a newly instituted series of scientific articles that provide evidence-based scientific opinions on topical and important issues in biomaterials science. They have some features of an invited editorial but are based on scientific facts, and some features of a review paper, without attempting to be comprehensive. These papers have been commissioned by the Editor-in-Chief and reviewed for factual, scientific content by referees.