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

proposed by Kokubo and his colleagues.

 

Chikara Ohtsuki, Ph. D.

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)

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

HPO42-

1.0

1.0

SO42-

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

Na+

K+

Mg2+

Ca2+

Cl-

HCO3-

HPO43-

SO42-

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

(b) Dissolution of chemicals
(c) Adjustment of pH
(d) Storage (e) Notes

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

K2HPO43H2O

Assay min. 99.0%, Nacalai tesque, Kyoto, Japan

0.228 g

#5

MgCl26H2O

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

K2HPO43H2O

0.228 g

0.456 g

0.342 g

0.684 g

#5

MgCl26H2O

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
4) 1.5SBF

5) Estimation of degree of supersaturation

6) Reviews

 

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