Cell culture medium as an alternative to conventional simulated body fluid

Abstract

Simulated body fluid (SBF) has been widely used for bioactivity assessment of biomaterials. Many different recipes have been developed, but there is still room for improvement. We have suggested the use of cell culture medium to overcome the drawbacks of conventional SBF. Compared with conventional SBF, cell culture medium is easy to prepare. The normal practices in cell culturing, such as filtering, can eliminate insoluble precipitates in the medium and incubation at 37 °C in an atmosphere with 5% CO₂ also better simulates the in vivo environment. After 4 days immersion in carbonate buffered Dulbecco’s modified Eagle’s medium (DMEM), precipitates were found to have formed on the surfaces of hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). In order to further verify the use of cell culture medium for SBF studies, we carried out computational thermodynamic and kinetic analyses of the precipitation reaction to reveal the effect of pH and ion concentrations on the driving force and nucleation rate of precipitation of different calcium phosphates (CaP). In general, a slight increase in pH of the cell culture medium from physiological pH (pH 7.4) would favor CaP precipitation thermodynamically and increase the rate, as in the case of r-SBF reported previously. [Ca] and [P] have more impact on precipitation compared with other ions, but the effect is consistent among different materials, indicating that other cell culture media with slightly different compositions may also be used. This study also shows that matching the buffer with the environment is required and fetal bovine serum (FBS) slows down surface CaP precipitation on HA.

Introduction

In 1991, Kokubo et al. proposed that the essential requirement for an artificial material to bond to living bone is the formation of bone-like apatite on its surface when implanted in the living body, and 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. They suggested that the in vivo bone bioactivity of a material can be predicted from the apatite formation on its surface in SBF. In 2006, Kokubo et al. reassessed the validity of SBF studies to predict bone-bonding ability using results from various groups. They concluded that the examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material. Thus, the number of animals used and the duration of animal experiments can be reduced remarkably by using this method [1], [2].

In 2009, Bohner and Lemaitre suggested the requirement of CO₂ partial pressure of 0.05 atm (5%) since human serum is in equilibrium with such a partial pressure [3]. Indeed, humidified atmosphere with 5% CO₂ is commonly used in cell culturing and this inspired us on the use of cell culture medium for SBF study. As the aim of SBF study is to predict in vivo bioactivity, it is better to simulate the in vivo environment as closely as possible and cell culture system in life science is a well established system to simulate the in vivo environment. Therefore, we proposed some modifications to typical in vitro cell culture assays on biomaterials in order to better simulate the physiological environment for our SBF study. The three easy-to-perform modifications are: 1) cells were not added; 2) the biomaterial surface area to medium volume ratio was smaller compared to cell culture assay to minimize changes in ion concentrations during immersion and 3) no serum was added as serum slows down the precipitation process at high concentration ([4] and also confirmed in this study). On the other hand, using plastic six well culture plates as the containers also allow easier handling of a large number of samples and plastic containers with smooth surface can avoid the possible apatite nucleation from the glass surface [2]. Furthermore, Bohner and Lemaitre suggested that there is still room for improvement in preparing SBF solutions in the following ways: (i) the procedure is long and tricky; (ii) the solutions are not filtered; (iii) the carbonate content is not controlled [3].

Our approach of SBF study avoided these described limitations as follows: (i) HEPES-free Dulbecco’s Modified Eagle Medium (DMEM), a cell culture medium, was used in this study. It is relatively easy to prepare and also commercially available, which would facilitate more reproducible results. (ii) The solution used in this study was filtered through a 0.22 μm filter (which is a common procedure to sterilize cell culture medium) to remove insoluble contaminants which can have a significant effect on precipitation. (iii) The medium was carbonate buffered as in the case of human blood and this study was performed in a 37 °C incubator under a humidified atmosphere of 95% air and 5% CO₂, which better simulates the in vivo environment. HEPES-free medium can be used in our study because of the controlled CO₂ level. Without controlled CO₂ level, the buffering capacity of the carbonate system would be affected.

In addition, cell culture medium contains essential components for cell culture. These include inorganic salts, vitamins, amino acids, etc. As most nutrients are supplied to cells by the blood circulation system, the ion concentrations in a cell culture medium should have high similarity to those of human blood plasma. In terms of simulating the in vivo environment, a cell culture medium can be a better choice as it also contains other components present in in vivo systems besides inorganic salts. It happened that Mandel and Tas have also noted the nutritional advantages of DMEM and suggested the use of HEPES buffered DMEM as an alternative to SBF in their study of brushite to octacalcium phosphate transformation using classical SBF procedures [5]. A brief summary of the ion concentrations in blood plasma, SBF solutions and DMEM are shown in Table 1.

In this study, we have demonstrated surface precipitate formation on CaP materials using carbonate buffered DMEM in 5% CO₂ atmosphere as the immersion medium, validating the use of it in SBF studies. Other cell culture media may also be used, depending on availability, in different laboratories, but they may differ slightly in ion concentrations. The ion concentrations of some commonly used cell culture media and physiological buffers are summarized in Table 2. In order to assess whether other cell culture media can be used, we carried out computational thermodynamic and kinetic analyses of different SBF components and different SBF recipes, including cell culture media and physiological buffers.