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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 bottles including 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 water well.
• 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 oC.
(b) Dissolution of chemicals
• Put 750 mL(=cm3) of ultra-pure water into a 1000 mL beaker (polyethylene beaker is preferred). Stir the water and keep its temperature at 36.5 oC with magnetic 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 solution in 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
• Calibrate 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 the temperature is at 36.5 oC. At this point, pH of the solution is approximately 7.5. Titrate 1kmol/dm3-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 solution, adjusting the total volume of the solution to 1000 mL, and the shake the flask well. Keep the flask at room temperature until its temperature should be approximately 20 oC. After cooling, add ultra-pure water again, the solution to the total volume of the solution to 1000 mL, and then shake the flask well.
(d) Storage
• Rinse a polyethylene (or polystyrene) bottle of 1000 mL with a bit of the prepared solution (SBF), at least three times. Transfer the solution from the flask to the polyethylene bottle.
• Store the bottle in a refrigerator at 5-10 oC.
(e) Notes
• Stability of the solution obtained must be examined. Put 50 mL of the solution in a polystyrene bottle and place it in incubator at 36.5 oC. After 2-3 days, check whether the solution has any precipitation or not. If any precipitation would be found, do not use the solution.
• Bottles in which precipitation occur must not be used for any further experiments, because some calcium phosphates would be adhered on their walls inside. A precipitation of calcium phosphate especially such as hydroxyapatite easily induces further formation of hydroxyapatite in the solution, since the simulated body fluid (SBF) is already supersaturated.
Table III Reagents for preparation of SBF (pH 7.25, 1 L).
Order Reagent Amount
#1 NaCl Assay min. 99.5%, Nacalai tesque, Kyoto, Japan 7.996 g
#2 NaHCO3 Assay (after drying) min. 99.5-100.3%, Nacalai tesque, Kyoto, Japan 0.350 g
#3 KCl Assay min. 99.5%, Nacalai tesque, Kyoto, Japan 0.224 g
#4 K2HPO4・3H2O Assay min. 99.0%, Nacalai tesque, Kyoto, Japan 0.228 g
#5 MgCl2・6H2O Assay min. 98.0%, Nacalai tesque, Kyoto, Japan 0.305 g
#6 1 kmol/m3 HCl 87.28 mL of 35,4% HCl is diluted to 1000 mL with volumetric flask 40 cm3
#7 CaCl2 Assay min. 95.0%, Nacalai tesque, Kyoto, Japan
Use after drying at 120 oC for more than 12 hours 0.278 g
#8 Na2SO4 Assay min. 99.0%, Nacalai tesque, Kyoto, Japan 0.071 g
#9 (CH2OH)3CNH2 Assay (after drying) min. 99.9%, Nacalai tesque, Kyoto, Japan 6.057 g
#10 1 kmol/m3 HCl See above Appropriate amount for adjusting pH

如果是模拟渗透压的话一般使用0.9%氯化钠溶液或者5%葡萄糖溶液