Rapid coating of Ti6Al4V at RT with a calcium phosphate solution similar to 10x synthetic body fluid
Journal of Materials Research, 19 (9), 2742-2749 (2004) ® PDF
This study, performed by Dr. A. Cuneyt Tas, reports the rapid formation of apatitic calcium phosphate coatings on Ti6Al4V using high ionic strength solutions compared to synthetic/simulated body fluids (SBFs).
The super-strength solution developed in this study has ten times the concentration of calcium and phosphate ions as compared to conventional SBF, and is referred to as 10 x SBF.
The idea is to significantly enhance the rate of coating formation, which requires 1 to 3 weeks of immersion time using conventional 1.5 X SBF.
The interesting features of the present technique are given in the following. First, the solutions do not need any buffering agents. Given the short duration of coating period, these are not really needed.
Second, during the process homogeneous precipitation of nano-clusters took place. However, their presence did not adversely affect the coating process.
Third, other than simple surface treatments to begin with, no other additional intermediate steps were necessary. The only step needed after the preparation of the solution from reagents is the addition of proper amounts of NaHCO3 just prior to the coating procedure.
Fourth, such a procedure led to a significant enhancement of coating rate enabling the formation in as little as 2 hours.
Finally, the adhesion strength of the coatings was comparable to coatings produced from 1.5 X SBF over a prolonged period of time.
SBF (synthetic/simulated body fluid) solutions are able [1-3] to induce apatitic calcium phosphate formation on metals, ceramics or polymers (with proper surface treatments) soaked in them.
SBF solutions, in close resemblance to the Hanks Balanced Salt Solution (HBSS) , are prepared with the aim of mimicking the ion concentrations present in the human plasma. It is noted that physiological HBSS solutions are also able to induce apatite formation on titanium .
To mimic physiological solutions, conventional SBFs (i.e., 1, 1.5, 2, or 5 X SBF) have relatively low calcium and phosphate ion concentrations, namely, 2.5 mM and 1.0 mM, respectively, for 1 X SBF . pH of SBF solutions was typically brought to the physiologic value of 7.4 by using buffers, such as TRIS  or HEPES . The buffering agent TRIS present in conventional SBF formulations, for instance, is reported  to form soluble complexes with several cations, including Ca2+, which further reduces the concentration of free Ca2+ ions available for coating.
The hydrogencarbonate ion (HCO3-) concentration in conventional SBF solutions was between 4.2 mM (equal to that of HBSS) and 27 mM [7, 9, 10].
However, having their ionic compositions similar to that of human blood plasma, SBF formulations have only been slightly supersaturated with respect to precipitation of the apatitic calcium phosphates. As a direct consequence, nucleation and precipitation of calcium phosphates from SBFs are rather slow . To get total surface coverage of a 10 x 10 x 1 mm titanium or titanium alloy substrate immersed into a 1.5x SBF solution, one typically needs to wait for 2 to 3 weeks, with frequent replenishment of the solution .
The motivation in this work is to enhance the kinetics of coating deposition. This enhanced kinetic should do away with the necessity of using buffers.
In order to achieve the above objective, Barrere et al. [13-17] have recently developed unique 5 X SBF-like solution recipes (with pH values close to 5.8), which did not employ any buffering agent, such as TRIS or HEPES. In these studies [13-17], coating was achieved by employing two different solutions (solutions A and B as they referred), and pH was adjusted by bubbling CO2 gas into the reaction chamber. A coating thickness of about 30 mm was achieved only after 6 h of immersion. However, they also introduced additional intermediate steps. These included  immersing the metal strips in the first solution (to seed the surface with calcium phosphate nuclei) for 24 h at 37°C, followed by another soaking in their second solution (to form the actual coat layers) for 6 to 48 h at 50°C . These additional intermediate steps add extra time and opposes the advantage gained by the enhanced kinetics.
The aim of this study was to present the preparation of a new acidic solution, which contains 10 times the calcium and phosphate ion concentrations of human blood plasma. Such a solution should enhance the kinetics of coating formation even more. Further it is preferred that other than the surface treatment step, not too many intermediate steps are involved. The only step that is needed is to add NaHCO3 into the solution to raise its pH to around 6.5.
The resultant solution is able to coat Ti6Al4V strips for the first time (RT: 22±1°C) rapidly, in as little as 2 hours. It is shown that it is not necessary to use biomimetic conditions for coating purposes.
Preparation of Ti6Al4V strips
Sheets of Ti6Al4V (Goodfellow) were cut into rectangular strips with typical dimensions of 10 x 10 x 0.20 mm and first abraded manually with a 1200-grit SiC paper. Strips were then cleaned with acetone (15 min), ethanol (15 min) and deionized water (rinsing), followed by etching each strip in 150 mL of a 5 M KOH solution at 60°C for 24 h, in a sealed glass bottle. Thoroughly rinsed (w/water) strips were finally heat-treated at 600°C for 1 h in Al2O3 boats, with heating and cooling rates of 3°C/min.
Solution preparation recipe (for a total aqueous volume of 2 L) is given below in Table 1. The chemicals given in Table 1 are added, in the order written, to 1900 mL of deionized water in a glass beaker of 3.5 L-capacity. Before the addition of the next chemical, the previous one was completely dissolved in water. After all the reagents were dissolved at RT, the solution was made up to 2 L by adding proper amount of water. This stable stock solution of pH value of 4.35-4.40 can be stored at RT, in a capped glass bottle, for several months without precipitation.
Reagent Order Amount (g) Concentration (mM)
NaCl 1 116.8860 1000
KCl 2 0.7456 5
CaCl2.2H2O 3 7.3508 25
MgCl2.6H2O 4 2.0330 5
NaH2PO4 5 2.3996 10
Just prior to coating a Ti6Al4V strip, a 200 mL portion of this stock solution was placed into a 250 mL-capacity glass beaker, and a proper amount of NaHCO3 powder was added to raise the hydrogencarbonate ion (HCO3-) concentration to 10 mM, under vigorous stirring. Following the rapid dissolution of the NaHCO3, the pH of the clear solution rose to 6.50 at RT. This solution (with an ionic strength of 1137.5 mM) was then transferred to a 250 mL-capacity glass bottle, which contained the Ti6Al4V strip inside, tightly capped and kept at RT for 2 to 6 hours during coating.
After the experiments were over, the strips were taken out of the solutions and rinsed with an ample supply of deionized water and ethanol, followed by drying in air. Samples were characterized by XRD (Model XDS 2000, Scintag Corp., Cu K(), FTIR (Bruker, ATR-FTIR), and SEM (Hitachi S-4700 in the secondary electron mode, acceleration voltage 5-15 kV). Platinum sputtering was used to render conductive surfaces that were necessary for the SEM investigations. In order to measure the thickness of the coat layers, the strips were tilted by 45 degrees and studied by SEM.
The chemical and thermal treatment of Ti6Al4V strips prior to the coating runs were mainly performed according to the previously published methods [6, 18, 19]. However, in our modification to the alkali treatment procedure, we have used 5 M KOH solution in lieu of 5 M NaOH. Figures 1a and 1b show the surface of 5 M KOH + 600°C-treated metal surface, and the aggregated rosettes seen on the surface belong to a potassium titanate phase of an approximate composition of K2Ti5O11.
Fig. 1a Surface of 5 M KOH + 600°C treated Ti6Al4V strips prior to coating at low magnification
Fig. 1b Surface of 5 M KOH + 600°C treated Ti6Al4V strips prior to coating at high magnification
A phase of similar stoichiometry (i.e., Na2Ti5O11) was also observed in case of using 5 M NaOH+600°C-treatment . The surface of the alkali- and heat-treated strips also contained rutile (TiO2) as a minor phase. K ions on the surfaces of such strips, when exposed to the coating solution, are released into the solution in exchange of H3O+ ions, and eventually resulting in the formation of a Ti-OH layer. Ca2+ ions from the coating solution are then incorporated in this basic layer and act as embryonic sites for the nucleation of carbonated apatitic calcium phosphates . The dimensions of crevices or pits created on Ti6Al4V surface in the etching step of 5 M KOH-soaking was found (Fig. 1) to be much larger than those created in using 5 M NaOH . Bigger crevices (as compared to sub-micron pits obtained with NaOH) are more suitable for the attachment of few microns-large calcium phosphate globules.
The coating solution described above was not stable against precipitation (at RT) after the addition of NaHCO3 to raise its pH to the vicinity of 6.5. The stability against homogeneous precipitation only lasted from 5 to 10 minutes at RT, following the addition of NaHCO3.
After that period, solutions containing the metal strips slowly started to display turbidity (from 10 minutes to the end of the first hour), and by the end of 2 hours the solution turned opaque. The colloidal precipitates formed in the solution stay suspended, and could only be separated from the mother liquor by centrifugal filtration (>3000 rpm).
However, it is interesting to note that the solution pH at the end of 2 hours of soaking period stayed the same or slightly increased to around 6.57 or 6.58 (® download the pH chart).That slight increase in pH was ascribed to the release of CO2 . A pH decrease would have been encountered during the formation of colloidal precipitates due to H+ release, but as it was reported previously such a pH drop was not always observed [14, 20].
In order to perform a run with a 6 hours-of-total-soaking time, the coating solution for the same strip was replenished twice with a new transparent solution (of pH = 6.5) at the end of each 2-hours segment. The start of precipitation indicated the stage where the solution reached supersaturation.
Figure 2 depicts the SEM photomicrographs of coated surfaces of Ti6Al4V strips as a function of coating time (1 to 6 h; Figs. 2a through 2d) at RT.
Fig. 2a 1 hour soaking at RT in 10xSBF
Fig. 2b 2 hours soaking at RT in 10xSBF
Fig. 2c 6 hours soaking at RT in 10xSBF (low magnification)
Fig. 2d 6 hours soaking at RT in 10xSBF (high magnification)
The observed globules of apatitic calcium phosphate were quite similar to the previously reported results relevant to biomimetic SBF coating, excepting that biomimetic conditions were not met here. High-magnification photomicrograph of Fig. 2d showed that the globules actually consisted of petal-like nanoclusters. The significant extent of surface coverage of the tivanium strip, in only 6 hours of coating, was exemplified in the macro-scale SEM picture depicted in Fig. 2c. Cracks seen in the micrographs of the coat layers were probably formed during the drying process of the coated samples . The adhesion of the coat layers was resistant to finger-nail scratch tests, and there was no difference in adhesion strength as compared to 1.5 X SBF-coated Ti6Al4V strips.
XRD data of the coated strips also confirmed the nature of these globules, as shown in Figure 3.
Fig. 3 XRD data collected directly from the coated strips (S: peaks of substrate) ® download image
The intermingling morphology of the colloidal precipitates obtained from a coating solution at the end of 2 hours was given in Figures 4a and 4b.
Fig. 4a Apatitic precipitates recovered from the coating solutions
Fig. 4b Apatitic precipitates recovered from the coating solutions
These precipitates were filtered from their mother liquor by centrifugation, washed three times with water, and once with ethanol, followed by drying at RT overnight. The SEM micrograph of Figures 4a and 4b, and the XRD data of these precipitates given in Fig. 5, as well as the FTIR data supplied in Fig. 6, also indicated that the nano-crystalline, bone-like apatitic calcium phosphate formed in the solution aggregated during filtration and drying.
Fig. 5 XRD data for the colloidal precipitates formed in coating solutions
Fig. 6 FTIR data of the colloidal precipitates formed in coating solutions
Bands of the O–H stretching and bending of H2O were seen at, respectively, 3440 and 1649 cm-1. Presence of carbonate groups was confirmed by the bands at 1490-1420 and 875 cm-1. PO4 bands were recorded at 570 and 603 (n4), 962 (n1), 1045 and 1096 (n3) cm-1. It is important to note that neither the precipitates themselves nor the coating layer (on Ti6Al4V strips) contained CaCO3 (calcite) .
Titanium or titanium alloy surfaces are coated with a carbonated and poorly-crystalline apatitic calcium phosphate layer to impart the metal surface a certain degree of in vivo bioactivity. If this is the sole aim, then there is no need to maintain the pH value of a coating solution exactly at the physiologic value of 7.4. This point has been successfully confirmed in the work of Barrere, et al. [13-17, 22]. One only needs to be aware of the delicate balance between the solution pH, HCO3- ion concentration and temperature in determining which phases will be soluble or not under a specific set of those conditions . On the other hand, the presence of TRIS or HEPES (added for the sole purpose of fixing the solution pH at around 7.4) in a SBF formulation simply retards the coating process to the level that in order to obtain a decent surface coverage one needs to wait for 1 or 3 weeks [6-8, 18].
Fast coating solutions, sometimes named as supersaturated calcification solutions (SCS) are not new either; for instance, the pioneering work of Wen, et al.  showed that even in a TRIS-buffered SCS solution it would be possible to form 16 mm-thick calcium phosphate coat layers in after 16 hours of immersion. More recently, Choi, et al.,  reported the RT coating (about 10 mm-thick in 24 hours) of nickel-titanium alloy surfaces by a simple SCS solution, which was not even buffered at the physiologic pH. The present paper corroborates these previous findings and reports further improvements.
It is known that an amorphous calcium phosphate (ACP) precursor is always present during the precipitation of apatitic calcium phosphates from the highly supersaturated solutions, such as the one used here . Posner, et al.  proposed that the process of ACP formation in solution involved the formation first of Ca9(PO4)6 clusters which then aggregated randomly to produce the larger spherical particles or globules (as seen in Figs. 2d and 4), with the intercluster space filled with water. Such clusters (with a diameter of about 9.5 Angstrom ), we believe, are the transient solution precursors to the formation of carbonated globules with the stoichiometry of Ca10-x(HPO4)x(PO4)6-x(OH)2-x, where x might be converging to 1 .
Onuma, et al.  have demonstrated, by using dynamic light scattering, the presence of calcium phosphate clusters from 0.7 to 1.0 nm in size in clear simulated body fluids. They reported that calcium phosphate clusters were present in SBF even when there was no precipitation. This was true after 5 months of storage at RT. The solution coating procedure described here probably triggered the hexagonal packing  of those nanoclusters to form apatitic calcium phosphates, just within the first 5 to 10 minutes, following the introduction of NaHCO3 to an otherwise acidic calcium phosphate solution.
One can also speculate besides the substrate coating procedure outlined here that these 10 X SBF solutions may also be used to produce carbonated, bone-like apatitic calcium phosphate powders . Following proper physiologic-temperature and pH granulation processing, such biomimetic powders may, for instance, lead to the manufacure of collagen-apatite composites or self-setting orthopedic cements of high resorbability.
The use of NaHCO3 with a concentrated (10 times of Ca2+ and HPO42- ion concentrations) synthetic body fluid-like solution of ionic strength of 1137.5 mM allowed the formation of an apatitic calcium phosphate layer on Ti6Al4V at room temperature within 2 to 4 hours.
The coating solutions of pH 6.5 did not necessitate the use of buffering agents.
The surfaces of the Ti6Al4V strips were chemically etched in 5 M KOH solution and thermally treated afterwards at 600°C, prior to soaking in 10 x SBF.
Formation of colloidal precipitates, within the solution, was observed during the first hour of soaking at RT, but apparently the presence of those fine precipitates did not hinder the coating process.
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