BIOMIMETIC PROCESSING

SBF (synthetic/simulated body fluid) is made popular by Prof. Tadashi Kokubo (J. Non-crystalline Solids, 120, 138-151, 1990). Prof. Kokubo has since published hundreds of profile-raising articles on SBF.

The original SBF is named as c-SBF, and this Tris-buffered solution has a HCO₃^- ion concentration of only 4.2 mM (in stark contrast to the human blood plasma value of 27 mM). The SBF solution of Kokubo (such as the one described in T. Kokubo and H. Takadama, Biomaterials, 27 (2006) 2907-2915) thus have a significant (HCO₃^-) bicarbonate ion-deficiency, casting a shadow on its ability to truly mimic the composition of the (inorganic) electrolyte of the human blood plasma.

Just to mention here, Hanks’ Balanced Salt Solution (HBSS) also has the same 4.2 mM bicarbonate ion concentration.

c-SBF solutions do also possess an excess of Cl^- ions (148 mM) in them, in comparison to the human blood plasma Cl^- concentration of only 103 mM.

c-SBF solutions of Kokubo were prepared by using K₂HPO₄∙3H₂O. The selection of dipotassium hydrogen phosphate trihydrate, as the phosphate source, was causing the above-mentioned deviations in c-SBF from the electrolyte concentration of blood plasma.

The use of Na₂HPO₄∙2H₂O, as shown below, in preparing the 50 mM Tris-buffered SBF solutions would easily help to increase the HCO₃^- concentration to 27 mM and to reduce the Cl^- concentration to 125 mM.

We have published, for the first time in 1999 (http://www.cuneyttas.com/haurea1.pdf), how to prepare a Tris-buffered and 27 mM HCO₃^—containing SBF solution, also having a significantly reduced Cl^- ion concentration (to 125 mM).

These solutions (27 mM HCO₃-Tris-SBF) are as easy to prepare as the c-SBF solutions.

27-Tris-SBF solutions have enhanced ability of inducing apatite-like calcium phosphate on ceramics, metals and polymers in comparison to that of c-SBF solution, due to their proper HCO₃^- concentration. Having said that, the formation of apatite-like calcium phosphate on ceramics, metals or polymers immersed into any SBF solution does NOT imply anything about the biological activity (i.e., bioactivity) of those materials in vivo. On the other hand, inorganic supersaturated CaP solutions, such as SBF which are devoid of any biological agents, such as biomolecules, proteins, growth factors and alike, cannot be used to predict the bioactivity of a synthetic material considered for in vivo implantation.

An implanted material will first come into contact with the patient’s blood during its implantation, by the surgeon(s), in the OR (operation room). Blood proteins, such as albumins (ALB), immunoglobulin (IgG) antibodies, fibronectin (FNT), fibrinogen (FGN) and high molecular weight kininogen

(HMK) will first try to attach themselves, in a specific succession, onto the available surface of that biomaterial (Reference: L. Vroman, Colloids and Surfaces B-Biointerfaces, 2008, Vol. 62, pp. 1-4). It is obvious that that biomaterial must possess enough surface area (in a laboratory setting, surface area is measured by the BET (Brunauer-Emmett-Teller) technique and reported with the units of “m²/g” for a non-liquid or non-gas material) in order to attach onto itself a significant amount of those blood proteins. (Human body contains more proteins beyond those present in blood, and this only serves to further complicate the situation.) If there would ever be a calcium phosphate nanoparticle nucleation/deposition process (e.g., in the case of implanting the material directly into a bony site) to in vivo take place on the surface of the implanted material, then the biological calcium phosphate nanoparticles may only form on the above-described layer (see Vroman’s article) of proteins. Therefore, to simply immerse materials in an SBF solution at 37°C, to observe the formation of CaP spherulites on their surfaces is more than a naïve approach in testing the so-called “bioactivity” of that material, in total negligence of biology and the role of proteins in crystallization in the human body. (Read: H. Pan, X. Zhao, B. W. Darvell, and Lu W. W., Acta Biomaterialia, 2010, Vol. 6, pp. 4181-4188)

If one heats a portion of an SBF solution (without having any immersed material in it) in a clean and inert container at 37°C for a couple of weeks, calcium phosphate spherulites will form autogenously. Therefore, under the light of the above experimental fact, keeping a certain material in the SBF solution and heating it at 37°C for a few weeks is NOT a test of bioactivity.

However, SBF is not useless, it can be positively used to increase the surface area of a material by such prolonged immersions at 37°C; during that immersion the surface of the material will accrue those CaP spherulites which shall have nanoneedles/nanowhiskers on their external boundaries; such nanoneedles help to efficiently increase the overall surface area of the immersed solid material. SBF is “a tool of processing the material prior to its implantation;” but not a tool for testing its in vitro bioactivity.

If the material immersed in the SBF solution has a certain chemical solubility, then some ions will dissolve from the material to the solution. For example, if the soaked material contains Ca²⁺ ions in its crystalline (or non-crystalline) structure and if these Ca²⁺ are not strongly bound to other ions (usually anions) of the given structure and if such Ca²⁺ ions leach into the solution, this will cause the Ca/P molar ratio (initially 2.50) of the SBF solution to slightly increase; this phenomenon will trigger the nucleation of CaP precipitates or spherulites. Such leaching/transfer of ions from the soaked material to the solution side will disturb the delicate ionic balance and ionic strength of the SBF and would cause it to precipitate nanoparticles of apatitic CaP. This event does not tell anything about the bioactivity of that immersed material, this is only a chemical dissolution process and is simply related to the local attainment of the solution supersaturation for the onset of the precipitation of apatitic CaP nuclei.

Moreover, if the immersed material has a basic surface (sometimes researchers achieve that basic surface by pre-soaking the material in strongly alkaline solutions, such as those of NaOH, KOH, and alike), then that basic surface at the material-solution interface would trigger the nucleation of nanoparticles of CaP. This is, again, not bioactivity. There is no biology-related activity in such inorganic nucleation phenomena.

A vibrant example to this kind of a basic surface accruing apatitic CaP precipitates/spherulites (i.e., on Teflon (PTFE) with a basic surface) is given by L. Grondahl, F. Cardona, K. Cheim, and E. Wentrup-Byrne, J. Mater. Sci. Mater. Med., 2003, Vol. 14, pp. 503-510. Should we understand the following now? “Teflon pre-treated in a strong base then placed into the SBF solution and formed carbonated apatitic CaP spherulites on its surface at the human body temperature, therefore, Teflon is a bioactive material.” This is an incorrect conclusion one my draw out of the experiment of Grondahl et al.

Please see the below articles via their weblinks on the preparation and uses of 27 mM HCO₃-Tris-SBF solutions, which mimic the human blood better than c-SBF solutions:

1.) J. Eur. Ceram. Soc., 19, 2573-2579 (1999)

2.) Biomaterials, 21, 1429-1438 (2000)

3.) J. Mater. Sci. Lett., 20, 401-403 (2001)

4.) J. Am. Ceram. Soc., 87, 2195-2200 (2004)

5.) J. Am. Ceram. Soc., 88, 3353-3360 (2005)

6.) J. Mater. Sci. Mater. Med., 17, 697-707 (2006)

7.) J. Biomed. Mater. Res., 78A, 481-490 (2006)

8.) Mater. Sci. Eng. C, 27, 432-440 (2007)

9.) J. Mater. Res., 22, 1593-1600 (2007)

10.) J. Am. Ceram. Soc., 90, 2358-2362 (2007)

11.) Mater. Sci. Eng. C, 28, 129-140 (2008)

12.) Acta Biomaterialia, 10, 1771-1792 (2014)

Ion Human Blood Plasma (mM) Tas-SBF (mM) Kokubo-SBF (mM)

Na⁺ 142 142 142

K⁺ 5 5 5

Mg²⁺ 1.5 1.5 1.5

Ca²⁺ 2.5 2.5 2.5

HPO₄^2- 1 1 1

HCO₃^- 27 27 4.2

Cl^- 103 125 147.8

SO₄^2- 0.5 0.5 0.5

Buffering agent -- Tris Tris

Preparation of 27 mM HCO₃-Tris-SBF (the below recipe has been described and published in the above articles)

Notes: (1) only use high purity deionized water (free of dissolved carbon dioxide, it is advised to boil the water just before using it, then cool it down to RT and store it in a sealed environment free of atmospheric carbon dioxide) and use chemicals of the highest possible purity your research budget can afford,

(2) do never use extremely hygroscopic, anhydrous CaCl₂ as the source of calcium,

(3) do not use K₂HPO₄

1000 mL-capacity glass beaker (use a hot-plate/magnetic stirrer); use a Teflon-coated magnetic stirrer

+ 960 mL deionized water

+ 6.5456 g NaCl

stir vigorously at RT for 3 min

+ 2.2682 g NaHCO₃

stir vigorously at RT for 3 min

+ 0.373 g KCl

stir vigorously at RT for 3 min

+ 0.1419 g Na₂HPO₄

stir vigorously at RT for 3 min

+ heat the solution to 36.5-37°C

+ 0.3049 g MgCl₂×6H₂O

stir vigorously for 3 min

+ 9 mL of 1 M HCl solution (use a pipette and add it slowly)

stir vigorously for 3 min

+ 0.3675 g CaCl₂×2H₂O

stir vigorously for 3 min

+ 0.071 g Na₂SO₄

stir vigorously for 3 min

+ 6.057 g Tris [= (CH₂OH)₃CNH₂]

Upon adding Tris, the solution should become turbid; keep stirring

+ insert the pH electrode into the solution, which should be at around 37°C until now

+ 30 mL of 1 M HCl (add slowly, in 5 mL portions, by using a pipette, your addition plan is 5 + 5 + 5 + 5 + 5 + 5 mL = 30 mL)

(with the addition of each 5 mL portion of 1 M HCl, pH will gradually drop towards 7.4)

+ keep stirring

+ be careful when adding the last and 6^th “5 mL portion” of 1 M HCl, add this 5 mL portion quite slowly (i.e., drop by drop) while watching the pH meter reading

+ you can easily adjust the pH at 7.4 (at 36.5 or 37°C)

+ make sure that pH = 7.4 @ 37°C (pH=7.38, 7.37, 7.41 or 7.39 are also OK)

+ measure the total volume of the transparent solution, if it is not exactly equal to 1000 mL, you must add deionized water to complete the volume to 1000 mL

+ always keep your SBF solution in a clean glass media bottle (of 1 L-capacity), tightly capped, in a refrigerator at +4° to 5°C (i.e., a regular refrigerator)

+ write the date of preparation on your glass bottle

+ do not use SBF solutions older than 30 to 40 days (since within that time period they will autogenously precipitate colloidal spherules of carbonated, apatitic calcium phosphate; sometimes those very fine precipitates may not be visible to the naked eye, but dynamic light scattering (DLS) will always be able to confirm the presence of such invisible (of course, to the naked eye) precipitates in both “new” and “old” SBF solutions)

When you repeat this preparation procedure at least two times, you will see that it is actually very easy!

+++ Perform the SBF experiments in clean, glass media bottles;

do not use plastic bottles, if you do so you may grow bacteria, because plastic surfaces are rough, consist of numerous microscopic crevices or protrusions; glass surfaces are smooth; SBF solutions at 37°C forms a suitable habitat for numerous bacteria to grow!

For different uses of 27 mM HCO₃-Tris-SBF solutions (in biomimetic coating or testing of metals, ceramics and polymers), you may visit the following weblinks:

http://www.cuneyttas.com/hasbf37-citat.htm

http://www.cuneyttas.com/haurea-citat.htm

http://www.cuneyttas.com/SBF-compare-citat.htm

http://www.cuneyttas.com/ha-enzyme-citat.htm

For learning more about what SBF solutions are or are not;

Contact: Prof. A. Cuneyt Tas, Ph.D. c_tas@hotmail.com www.cuneyttas.com

For a better SBF solution which perfectly mimics the human blood plasma in terms of its inorganic ion concentrations, and for an SBF solution completely free of 50 mM Tris or 50 mM Hepes, visit : http://www.cuneyttas.com/How-to-prepare-SBF-solution-with-lactic-acid.htm