Introduction Kokubo and his colleagues developed an acellular simulated body fluid that has inorganic ion concentrations similar to those of human extracellular fluid, in order to reproduce formation of apatite on bioactive materials in vitro. This fluid can be used for not onlyevaluation of bioactivity of artificial materials in vitro, but also coating of apatite on various materials under biomimetic conditions. The simulated body fluid is often abbreviated as SBF or Kokubo solution. The ion concentrations of SBF are given on Table I.
Table I Ion Na + K+ Mg 2+ Ca 2+ Cl HCO 3 HPO 4 2SO4 2-
Ion concentrations of the simulated body fluid and human blood plasma. Humanblood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
Concentration (mmol/dm3 ) Simulated body fluid (SBF) 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5
The pH of SBF is adjusted to pH 7.25 at 36.5 o C, by using 50 mM (=mmol/dm 3 ) of tris(hydroxymethyl)aminomethane and approximately 45 mM of HCl. When apatite-forming ability of the specimen is not so high, pH of SBF is sometimes adjusted to pH 7.40.Modification of ion concentrations is available to SBF. Some types of the modification are given on Table II. Table II Solution 1.5SBF SBF(7.5) SBF Na + 213.0 142.0 142.0 K+ 7.5 5.0 5.0 Modification of the ion concentrations in SBF. Concentrations (mol/m 3 ) Mg 2+ 2.3 1.5 1.5 Ca 2+ 3.8 2.5 2.5 Cl 221.7 147.8 147.8 HCO 3 6.3 4.2 4.2 HPO 4 31.5 1.0 1.0 SO4 20.8 0.5 0.5 pH 7.25 7.50 7.25 Buffering agent A BB
Buffer A: (CH 2 OH) 3 (CNH) 2 75 mol/m3 , appropriate amount (app. 67.5 mol/m3 ) of HCl. Buffer B: (CH 2 OH) 3 (CNH) 2 50 mol/m3 , appropriate amount (app. 45 mol/m3 ) of HCl.
Preparation of SBF SBF is a metastable solution containing calcium and phosphate ions already supersaturated with respect to the apatite. Therefore SBF is prepared as follows.: (a) Cleaning Clean all the bottlesincluding flasks, beakers etc. with dilute hydrochloric acid solution, sterilizing agent and ultra-pure water in this order Immerse all the bottles etc. in dilute hydrochloric acid solution for several hours. Remove the bolles from the solution and wash with tap water well. Immerse the bottles etc. in sterilizing liquid for overnight. Remove thme from the liquid, and washed with ultra-pure waterwell. Wash the bottles with ion-exchanged water for several times and cover their mouths with wrapping film. The bottles do not need to be dried. If the bottles would need to be dried place them in drier below 50 o C. (b) Dissolution of chemicals Put 750 mL(=cm 3 ) of ultra-pure water into a 1000 mL beaker (polyethylene beaker is preferred). Stir the water and keep its temperature at 36.5 o C withmagnetic stir with heater. The beaker is preferred to be placed in clean bench, to avoid dusts. Add each chemical given in Table III into the water until #8, one by one in the order given in Table III, after each reagent was completely dissolved. Weigh a chemical with weighing bottle. Add it in the water. Wash the remaining chemical on the weighing bottle with ultra-pure water and add the solutionin the water. Addition of reagent #9 should be little by little with less than about 1g, in order to avoid local increase in pH of the solution. (c) Adjustment of pH Calbrate the pH meter with fresh standard buffer solution. After #9 on the order in Table III, check the temperature of the solution in the beaker, and place the electrode of pH meter in the solution. Measure its pH while thetemperature is at 36.5 o C. At this point, pH of the solution is approximately 7.5. Titrate 1kmol/dm 3 -HCl solution with pipette to adjust the pH at 7.25 (or 7.40). After the adjustment of pH, transfer the solution from the beaker to a glass volumetric flask of 1000 mL. Wash the inside of the beaker with ultra-pure water several times and add the solution to the flask. Add ultra pure water to the...