Index.html

How to prepare the simulated body fluid (SBF) and its related solutions,

proposed by Kokubo and his colleagues.

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 only evaluation 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 concentrations of the simulated body fluid and human blood plasma.

Ion	Concentration (mmol/dm³)
Ion	Simulated body fluid (SBF)	Human blood plasma
Na⁺	142.0	142.0
K⁺	5.0	5.0
Mg²⁺	1.5	1.5
Ca²⁺	2.5	2.5
Cl^-	147.8	103.0
HCO₃^-	4.2	27.0
HPO₄^2-	1.0	1.0
SO₄^2-	0.5	0.5

The pH of SBF is adjusted to pH 7.25 at 36.5 ^oC, by using 50 mM (=mmol/dm³) 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

Modification of the ion concentrations in SBF.

Solution	Concentrations (mol/m³)								pH	Buffering agent
Solution	Na⁺	K⁺	Mg²⁺	Ca²⁺	Cl^-	HCO₃^-	HPO₄^3-	SO₄^2-	pH	Buffering agent
1.5SBF	213.0	7.5	2.3	3.8	221.7	6.3	1.5	0.8	7.25	A
SBF(7.5)	142.0	5.0	1.5	2.5	147.8	4.2	1.0	0.5	7.50	B
SBF	142.0	5.0	1.5	2.5	147.8	4.2	1.0	0.5	7.25	B

Buffer A: (CH₂OH)₃(CNH)₂ 75 mol/m³, appropriate amount (app. 67.5 mol/m³) of HCl.
Buffer B: (CH₂OH)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 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.

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 the temperature is at 36.5 ^oC. At this point, pH of the solution is approximately 7.5. Titrate 1 kmol/m³-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

Appendix

A solution with ion concentrations 1.5 times of SBF (1.5SBF) can be prepared, in order to accelerate the apatite formation.

Table IV

Amounts of Reagents for preparation of SBF and 1.5SBF.

Order	Reagent	Amount
		SBF		1.5SBF
		1000 mL	2000 mL	1000 mL	2000 mL
#0	Ultra-pure water	750 mL	1500 mL	750 mL	1500 mL
#1	NaCl	7.996 g	15.992 g	11.994 g	23.988 g
#2	NaHCO₃	0.350 g	0.700 g	0.525 g	1.050 g
#3	KCl	0.224 g	0.448 g	0.336 g	0.672 g
#4	K₂HPO₄・3H₂O	0.228 g	0.456 g	0.342 g	0.684 g
#5	MgCl₂・6H₂O	0.305 g	0.610 g	0.458 g	0.916 g
#6	1 kmol/m³ HCl	40 cm³	80 cm³	60 cm³	120 cm³
#7	CaCl₂	0.278 g	0.556 g	0.417 g	0.834 g
#8	Na₂SO₄	0.071 g	0.142 g	0.107 g	0.214 g
#9	(CH₂OH)₃CNH₂	6.057 g	12.114 g	9.086 g	18.172 g
#10	1 kmol/m³ HCl	Appropriate amount for adjusting pH

References

1) Prototype-SBF

T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, "Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W", J. Biomed. Mater. Res., 24, 721-734 (1990).
T. Kokubo, S. Ito, Z. T. Huang, T. Hayashi, S. Sakka, T. Kitsugi and T. Yamamuro, "Ca,P-rich layer formed on high-strength bioactive glass-ceramic A-W", J. Biomed. Mater. Res., 24, 331-343 (1990).

2) Conventional-SBF

C. Ohtsuki, H. Kushitani, T. Kokubo, S. Kotani and T. Yamamuro, "Apatite formation on the surface of Ceravital-type glass-ceramic in the body", J. Biomed. Mater. Res., 25, 1363-1370 (1991).
S.-B. Cho, Kazuki Nakanishi, T. Kokubo, N. Soga, C. Ohtsuki, T. Nakamura, T. Kitsugi and T. Yamamuro, "Dependence of Apatite Formation on silica Gel on Its Structure: Effect of Heat Treatment", J. Am. Ceram. Soc., 78 [7], 1769-1774 (1995).
C. Ohtsuki, Y. Aoki, T. Kokubo, Y. Bando, M. Neo and T. Nakamura, "Transmission electron microscopic observation of glass-ceramic A-W and apatite layer formed on its surface in a simulated body fluid", J. Ceram. Soc. Japan, 103 [5], 449-454 (1995).

3) Modification of SBF

C. Ohtsuki, T. Kokubo, M. Neo, S. Kotani, T. Yamamuro, T. Nakamura and Y. Bando, "Bone-Bonding Mechanism of Sintered b-3CaO^.P₂O₅", Phosphorus Research Bulletin, 1, 191-196 (1991).
A. Oyane, H.-M. Kim, T. Furuya, T. Kokubo, T. Miyazaki and T. Nakamura, "Preparation and assessment of revised simulated body fluid", J. Biomed. Mater. Res., 65A, 188-195 (2003).

4) 1.5SBF

M. Tanahashi, T. Yao, T. Kokubo, M. Minoda, T. Miyamato, T. Nakamura and T. Yamamuro, "Apatite coating on organic polymers by a biomimetic process" J. Am. Ceram. Soc., 77, 2805-2808 (1994).
Y. Abe, M. Kawashita, T. Kokubo and T. Nakamura, "Effects of Solutions on Apatite Formation on Substrate in Biomimetic Process", J. Ceram. Soc. Japan, 109 [2], 106-109 (2001).

5) Estimation of degree of supersaturation

Chikara Ohtsuki, Tadashi Kokubo and Takao Yamamuro, "Mechanism of apatite formation on CaO-SiO₂-P₂O₅ glasses in a simulated body fluid", J. Non-Crystl. Solids, 143, 84-92 (1992).

6) Reviews

Masao Tanihara, Toshiki Miyazaki, Shin-ichi Ogata and Chikara Ohtsuki, "Design and Development of Functional Biocompatible Hybrid Materials for Medical Applications", in Handbook of Organic-Inorganic Hybrid Materials and Nanocomposites, Vol. 2, ed. by H. S. Nalwa, American Scientific Publishers, 2003, pp. 265-293.
Chikara Ohtsuki, Masanobu Kamitakahara and Toshiki Miyazaki, "Coating bone-like apatite onto organic substrates using solutions mimicking body fluid", Journal of Tissue Engineering and Regenerative Medicine, 1 [1], 33-38 (2007).

---------------------------------------------------------

Please visit alternative site for preparing the SBF.