After the vertical immersion of SAHPC in 1.5 × Sr-SBF, or 1.5 × Zn-SBF for two successive soaking periods, i.e., 4 days, an apparent resorption of all the peaks was observed (Figs. 1, 2). All peaks were within the same positions as that of mother sample [24]. In general, the optical density of all the peaks in Sr-c-SBF, (Fig. 1), exceeded those in Zn-c-SBF, (Fig. 2), proving a limited range of resorption in the former with respect to the latter (Tables 3 and 4). A more specific difference was the behavior of the peak value of the “C-O” stretch ν₃ mode. It settled around the same value as that of SAHPC in Sr-c-SBF discs. While in Zn-c-SBF, it showed greater decrement.

Fig. (1) DRIFTS of Sr-c-SBF post 2nd, 3rd, 4th and 5th soaking periods in 1.5 X Sr-c-SBF. “S.” accounts for the area assigned to Strontianite, SrCO3

Fig. (2) DRIFTS of Zn-c-SBF post 2nd, 3rd, 4th and 5th soaking periods in 1.5 X Zn-c-SBF. “S.” accounts for the area assigned to Smithsonite, ZnCO3

Table 3

Progress in the Optical Density Values of the Assignment Groups on Sr-c-SBF, Measured According to Kubelka-Munk Function

Table 4

The Progress in the Optical Density Values of the Assignment Groups on Zn-c-SBF, Measured According to Kubelka- Munk Function

A “built in” behavior is shown. This is precisely true for the peaks assigned for OH^- stretching vibration and PO₄^3- stretching and bending vibration modes. This growth, whose higher values for Sr-c-SBF than that of Zn-c-SBF, indicates the incorporation of water molecules which accompany the growth of inorganic calcium phosphate phases [14, 28-37]. There was a slight difference detected with respect to the behavior of “out-of-plane” C-O bending vibration ν₂ band. The band at 871 cm^-1 in Sr-c-SBF, continued its value loss, whereas, at 873 cm^-1 in Zn-c-SBF, it showed a slight increment. Besides, peaks at 712 cm^-1, (Sr-c-SBF), and at 715cm^-1, (Zn-c-SBF) that are assigned to “in-plane” C-O bending vibration, both showed slight decrement. Such peaks as a whole assure the presence of inorganic CO₃^2- group [16,21,31-33,36]. In general, the carbonate content at this point showed slight drop on Sr-c-SBF, which was actually as triple in value as that of Zn-c-SBF. In the latter, it stayed around the same values as that measured after 4 days soaking. This indicates a start of the alternative deposition of calcium and phosphate ions in the route of HA formation. The surfaces, at the moment, had already dissolved the extra content of the calcium carbonate phase that was deposited after the fast precalcification step [24]. Being ready for proper calcium phosphate deposition on a calcium rich surface, negatively charged phosphate ions started to show their contribution. Another difference is stated in the presence of the doublet due to the presence of a band around 712 cm^-1 and 623 cm^-1. This doublet characterizes the growth of aragonite (CaCO₃) [38]. Strontianite, SrCO₃, is one of the associated minerals to aragonite. In fact, Sr was reported to substitute Ca in aragonite up to 7500 part/million up to 40% [38]. This in turn explains the stability of carbonate content on Sr-c-SBF which should have been consumed in SrCO₃ growth. In a similar manner, this doublet indicates the growth of smithsonite, ZnCO₃. It is found as a secondary mineral in the oxidization zone of zinc ore deposits and in sedimentary deposits as a direct oxidization product of sphalerite. Zn can also substitute Ca in aragonite but not as efficiently as Sr [39].

The resultant charts showed growth and more differentiation for all the peaks. For both treatments, the water content with the resultant phase had undergone an abrupt increase. In contrast to Zn-c-SBF, it was time of carbonate content inhibition on Sr-c-SBF surface. Nevertheless, the overall value in the former was lower than that in the latter. This was accompanied by growth of phosphate content. The band assigned for PO₄^3- stretching vibration ν₃ was as twice in value as that shown post six days soaking. This was true for both treatments. These simultaneous events may be a step in the way of phosphate substitution to the carbonate in the resultant apatite coating. At this moment, it was not clear whether there were sites characteristic for strontianite and smithsonite on Sr-c-SBF and Zn-c- SBF respectively. This is because at these sites two highly differentiated peaks at 667 cm^-1, on Sr-c-SBF, and at 654 cm^-1, on Zn-c-SBF, assigned for OH^- bending vibration, were associated with others at 598 cm^-1, on the former, and at 593 cm^-1, on the latter, assigned for PO₄^3- bending vibration ν₄. This subsequently confirms the development of the apatite layer on the surface. Because the coating procedure was not vacuum sealed, peak appeared on both charts around 3108 cm^-1, which is assigned for C-H bond. Here, it is clear how Sr incorporation accelerated the growth of the apatite in comparison to Zn.

Stability, in the values of all peaks shown in these charts, is accompanied by a change in the carbonate to phosphate ratio with excess for the latter (Tables 3 and 4). The carbonate content here is ascribed to B-type which substitutes for phosphate groups rather than hydroxyl ones [34]. The sites assigned for strontianite or smithsonite continued their loss. This was accompanied by the decrement of carbonate content and increment in water and hydroxyl content. This in turn claims that the grown phase is carbonated apatite [14,33,40]. Consequently, Sr and Zn could be responsible for induceding growth of this phase. The difference here lies in the mechanism and activity of incorporation. Sr induced faster growth by keeping the carbonate content and induced apatite formation by direct phosphate substitution. While Zn, just dropped down the carbonate content and then induced the mutual incorporation of both phosphate and carbonate groups. In comparison to our previous study concerning the conventional biomimetic coating [24], the incorporation of both Sr and Zn induced the growth of apatite with higher carbonate content. This is better from a biological point of view because the biological apatite is not highly crystalline hydroxyapatite. It is poorly crystallized calcium-deficient as a result of various substitutions at regular HA lattice points. This is explained in details elsewhere [41].

The variation of both Ca⁺² and iP, uptake onto the samples is given in Fig. (3). It is here assumed that any change by decrease/increase in calcium or phosphorus concentrations, with respect to mother solution, would represent a deposition/release on/from Sr-c-SBF or Zn-c-SBF surfaces respectively. In 1.5 × Sr-c-SBF solution, the alternative behavior of Ca⁺ vs iP through successive soaking periods showed a deposition behavior. This agreed with the previously explained IR analyses, where, Sr-c-SBF kept the relatively high carbonate content in the initial periods and started earlier (than Zn-c-SBF) the phosphate deposition (Fig. 1). This explains why Ca⁺² showed in the soaking solutions higher values, indicating their release, followed by lower ones, indicating their deposition and contribution on the precalcified Sr-c-SBF surfaces [24]. On the other hand, an opposite trend was adopted in 1.5 × Zn-c-SBF. Ca⁺² started its deposition within the first soaking periods. This was synchronized by the release of the carbonate content as shown in IR analyses, (Fig. 2). When the carbonate content started to show some increment on Zn-c-SBF, starting from the third soaking period, a release behavior was recorded for Ca⁺². The ideal alternative Ca⁺² - iP behavior is not shown in 1.5 × Zn-c-SBF [38].

Fig. (3) Uptake of Ca+2 & iP from 1.5×Sr-SBF and 1.5 × Zn-SBF post each soaking periods with respect to the mother solution. (Where, Ca+2m & iPm are for mother solution, and, Ca+2n &iPn are specific for each period). The drawn curves represent the polynomial fitting of the third order to the obtained results.

To visualize the exact influence of incorporation of Sr⁺² on the Ca⁺² behavior, a study of the variation of percentage fraction of Ca⁺²vs Sr⁺² through the successive soaking periods is shown (Fig. 4). The calculations were done with respect to the mother solution using atomic absorption spectroscopy. The diagram represents the values such that if they are lower than 100%, a decrement in ionic concentration is recorded, otherwise, there is an increment with respect to the mother solutions. As shown in the diagram, Ca⁺² and Sr⁺² behave oppositely. This accounts to the approximately similar ionic radii of Sr⁺² (1.13Ả) and Ca⁺² (0.99 Ả). This confirms our claim (explaining the IR charts) of the substitutional competition in the grown coating layer between both ions, consequently, giving an evidence of the contribution of Sr⁺² to the resultant biomimetic surface. Also, this shows quite “step-wise” agreement with IR data, (Fig. 1) in which it was apparent that the peaks that initially appeared on Sr-c-SBF, assigned for strontianite disappeared after the last soaking periods.

Fig. (4) Variation of the percentage fraction of Ca+2vs Sr+2 post each soaking periods in 1.5 × Sr-c-SBF. The drawn curves represent the polynomial fitting of the second order to the obtained results.

The Ca/Sr competition is not really shown in case of Zn⁺² (Fig. 5). It started after about half the whole soaking procedure. From another point of view, this also indicates the interstitial incorporation of Zn⁺² into the grown apatite rather than the substitutional one. This is crystallographically true with respect to the ionic radius of Zn⁺² (0.83A˚). Besides, this gives the other side of the story of the disappearance of the peaks assigned for smithsonite during the progression of the soaking procedure, namely, in the second half of the process.

Fig. (5) Variation of percentage fraction of Ca+2vs Zn+2 post each soaking periods in 1.5 × Zn-SBF. The drawn curve represents the polynomial fitting of the second order to the obtained results.

Conductivity measurement accounts to ionic concentrations of designed soaking solutions. In fact, the decrease in the ionic concentration, by their uptake from solution and deposition onto the sample surface, would decrease the conductivity of the solution as a whole and vice versa. This would be represented as an “increased” decrement, in the following diagram (Fig. 6). For both solutions, the decrement slowed down gradually through the successive soaking periods. Although there was a starting resorption of the precalcified surface (shown earlier) which should have resulted in an overall increase in ionic concentration of the soaking solutions; this was compensated by the rapid uptake from SBFs. Besides, because we adopted a replenishment procedure of the soaking solutions, i.e., dynamic coating, this uptake decreased through the whole procedure with respect to the mother solution. The decrement of 1.5 x Sr-c-SBF was higher than that of 1.5 x Zn-c-SBF. This consequently shows the superiority of Sr⁺² over Zn⁺². After six days soaking, which corresponded to carbonate/phosphate substitution on Sr-c-SBF (Fig. 1), the conductivity started its increase, indicating dissolution. But, this was slightly changed after the fifth soaking period, which corresponded to more progression and differentiation of the phosphate bands on Sr-c-SBF. The conductivity trend showed a start of a new cycle of deposition (after 10 days soaking) and therefore, assures the continuity of the apatite growth. Therefore, Sr-c-SBF showed a desiring attitude for more depositions confirming, its bone seeking behavior [44]. In contrast, 1.5 X Zn-c-SBF continued overall increase in the ionic concentration in the last soaking periods, indicating an overall dissolution mechanism.

Fig. (6) Decrement in electrical conductivity of 1.5 × Sr-SBF (grey) and 1.5 × Zn-SBF (black) through various soaking periods with respect to the mother solution. (Where σm is the conductivity of the mother solution and σn is the conductivity of respective time interval). The drawn curve represents the polynomial fitting of fourth order to the obtained results.

Ca⁺² and iP ionic concentrations (Table 5) of soaking solutions interpret the real situation of apatite deposition. The surface was initially calcium rich due to pre-calcification [24]. The positively charged surface started to attract the negative phosphate ions. This was followed by the formation of a calcium poor, negatively charged surface that once again attracted the positive divalent calcium ions from the SBF. Consequently, an amorphous calcium phosphate phase was formed on the alloy surface. This in turn had undergone more deposition accompanied by selective dissolutions to reach the real poorly crystallized apatite that mimics the biological apatite. An important measurement would be the ionic product, Q_sp. (Fig. 7) illustrates the decrement of [Ca⁺²+Sr⁺²][iP] and [Ca⁺²+Zn⁺²][iP] through the successive soaking periods with respect to their mother solutions. The higher the values’ positivity, the higher the decrement in the ionic product. 1.5 X Sr-c-SBF showed lower decrement than that of 1.5 x Zn-c-SBF (Fig. 7). Therefore, the supersaturation level was kept at higher values for the former than the latter. According to the non classical theory of homogenous nucleation, indirect rapid and higher crystallization of amorphous calcium phosphates would be expected on Sr-c-SBF compared to Zn-c-SBF. In great resemblance, these results are in a general consistent with those of electrical conductivity (Fig. 6).

Table 5

Measured Values of Ca⁺², Sr⁺², Zn⁺², iP and their Total Ratio in Modified Sr and Zn Containing SBF

Fig. (7) The decrement of [Ca+2+Sr+2][iP], black, and [Ca+2+Zn+2][iP], red, post each soaking period with respect to their mother solutions. The polynomial fitting here is of the fourth order.

Compared to control treatment [24], Sr⁺² and Zn⁺² retarded the growth of highly dense apatite phase on Ti-6Al-4V, especially for the former. They also increased the calcium deficiency in the resultant phase. This can be easily explained through the assessment of the values of Ca⁺²+Sr⁺²/iP and Ca⁺²+Zn⁺²/iP (Table 5). Chou et al. [42] indicated that amorphous calcium phosphate, ACP, (25% crystallinity) showed a higher biosolubility than standard HA (63% crystallinity) and the lower crystallinity might be favorable to cell attachment. However, high biosolubility of ACP resulted in an increased pH of culture medium up to 8.2, which inhibited the proliferation of attached cells. Therefore, a balance between the phase structure (crystallinity) and its biosolubility should be considered.