3.1. SEM and EDS after Immersion in DMEM, SBF, and PBS
It is well documented that both biomineralization and biodegradation processes/phenomena occur either simultaneously and/or in competition with each other due to chemical reactivity [54
]. Thus, based on all of these, a bioactive surface will slightly dissolve in time, favoring the formation of a new apatite layer and promoting the formation of chemical bonds directly on the hard tissue [57
]. Meanwhile, through biodegradation, regardless of the mechanism that leads to the material removal/resorption, the tissue will grow directly in all the irregularities found on the surface without it being necessary for the interface to interact directly with the material [25
SEM images of each sample after its exposure in DMEM, SBF, and PBS can be observed in Figure 2
. In order to show details of the mechanism of biomineralization/degradation, the EDS results are also presented in Figure 2
Note that the coatings before biomineralization tests exhibited a Ca/P ratio of about 1.67 ± 0.02. The SEM images of surfaces before biomineralization are published in [34
]. Thorough characterization of the HAp and SiC-doped HAp coatings in terms of phase composition, topography and surface texture, adhesion between the coating and substrate, chemical bonds, and mechanical properties (hardness and elastic modulus) can be found in previous publications [33
]. Due to the dissolution/precipitation mechanism that occurs at the interface between HAp + SiC and the testing media, the Ca/P ratio of the HAp + SiC coating after immersion presented modifications which sustained the degradation and/or led to biomineralization.
According to the SEM images, the coated surfaces immersed in PBS suffered a degradation process that was initiated at the beginning of the tested 21 days. It is necessary to specify that in the literature the apatite formation mechanism starts with the partial dissolution of calcium ions from HAp, which then react with phosphate ions in PBS to form apatite [68
This result is also confirmed by the EDS analysis, which showed that the Ca/P ratio decreased by increasing the immersion time. From this it can be assumed that the dissolution process is predominant and not balanced by the apatite precipitation. It is worth mentioning that PBS is a balanced salt solution commonly used in biological research due to its resemblance to human extracellular fluid, and it has also been used in degradation studies [70
In SBF media, the developed layer also presented signs indicating partial degradation. For SBF acellular media, the EDS results indicated a Ca/P ratio above 2, suggesting the formation of a layer consisting of Ca-enriched hydroxyapatite. The SEM and EDS results of the coatings immersed in DMEM clearly showed the formation of new apatite phases, which increased with increasing the immersion time. Moreover, the chemical composition of the media influenced the HAp + SiC behavior. This was more visible for DMEM which, besides its similar ion concentrations with SBF, contains amino acids, vitamins, and other components that favor the precipitation of new apatite phases. However, further studies need to be performed in order to provide a wider perspective.
In the light of the aforementioned observations, it can be assumed that the HAp + SiC coatings presented some areas that are susceptible to the dissolution process, leading to their degradation, while other areas remained stable, as it can be seen in Figure 2
In PBS, the coatings started their degradation from the first day of immersion, initially presenting signs of cracks followed by areas of delamination from the substrate surface. Starting on day 14, larger areas without coating could be observed, indicating that the coating was more or less dissolved by the media. Overall it can be said that the coatings were more affected by the PBS and SBF solutions than by DMEM.
3.2. Mass Evolution in DMEM, SBF, and PBS
The mass evolution of the coatings immersed in DMEM, SBF, and PBS over 1, 3, 7, 14, and 21 days is presented in Figure 3
. It is generally known that the newly formed apatite layer is accepted as a hallmark of the bioactive materials and it is assumed to enable the quick formation of a mechanically stable and functional interface at the biomaterial–tissue level.
For the coatings immersed in SBF and PBS, the lost mass increased with increasing the immersion time, signifying that the coatings were degraded in these two media. Regarding the coatings immersed in DMEM, the mass increased with the immersion time, demonstrating good mineralization abilities. This effect was more obvious after 21 days of immersion. With respect to the acellular media used in this study, it can be said that DMEM presents not only different types of salts but also vitamins, proteins, and other nutrients, while SBF and PBS acellular media present some similarities in terms of their chemical compositions. Nevertheless, Porter et al. [74
] hypothesized that the different behavior of HAp + SiC could also be associated with the number and types of structural defects present in the coatings. They suggested that a higher number of defects could lead to an increased solubility and, consequently, a more elevated bone apposition rate, following a process of dissolution/precipitation [31
All things considered, it is possible that due to the preparation method, the developed coatings may have presented some compactness defects, thus influencing the films’ behavior in the acellular media. All together, these results suggest that in the presence of proteins, a higher interaction at the HAp + SiC and acellular media level is observed, thus limiting the coating degradation. According to a study by Schwarz, Si can bind to connective tissue and its components, specifically glycosaminoglycans, polysaccharides, and mucopolysaccharides [75
], although the mechanism by which this occurs is yet to be elucidated. Moreover, Hench et al. [76
] suggested that such materials have been shown to present higher bonding abilities to bone compared to their counterparts without Si by the spontaneous formation of a biologically active apatite-like layer on their surface.
3.3. XRD after Immersion in DMEM, SBF, and PBS
presents the XRD diffractograms of HAp + SiC compared to HAp before their immersion in the testing media. As can be seen, the characteristic peaks according to ICDD#09-0432 standard are present in the HAp structure and no other secondary phase was detected. It can be observed that the HAp XRD pattern is distorted. This finding is probably due to the Si peak from the substrate (Si-ICDD#04-002-0118 standard). In the XRD pattern of undoped HAp, there is a peak located around 10.8° with high intensity which can also be attributed to the brushite phase. It is well known that the acquisition of an XRD diffractogram often favors preferential crystal orientations, especially the phases with highly anisotropic morphologies such as brushite. In most cases, experimental XRD peaks may be significantly different compared to calculated ones, by variation in peak intensities. Drouet demonstrated that the (020) experimental peak becomes more intense than expected for randomly oriented crystals for brushite [77
]. He also reported that these modifications will separate brushite from the apatite compounds. Based on this published conclusion, we believe that it is possible that other CaP phases such as brushite may form around 10.8°. To sustain this state, a more complex analysis should be conducted, which is a further aim of our research.
With the addition of SiC, the XRD diffractogram changed its pattern and not all HAp peaks were detected, a fact which suggests that the coating tends towards amorphization due to the increase of nucleation, which leads to the reduction of grain size. Overall, SiC addition reduced the crystallinity of HAp and one peak characteristic for SiC and HAp was detected by XRD analysis. Moreover, it is clear that with SiC addition, the HAp peak observed in the HAp + SiC coatings shifted towards lower angles. This indicates that some structural changes, such as HAp elemental cell deformation, occurred. These findings are in good correlation with the findings of other researchers [78
XRD diffraction patterns of the coated surfaces after immersion in DMEM, SBF, and PBS are presented in Figure 5
. For the samples tested in DMEM, a broad line at the beginning can be observed, which could be a sign of an amorphous phase formation. Moreover, the line located at 63.6°, attributed to hydroxyapatite, is diminished with increasing the immersion time, suggesting a controlled release.
For samples immersed in SBF, the lines of hydroxyapatite overlap those of the bare substrate, making it difficult to state the presence or absence of apatite phases. After 3 days of immersion, some peaks found at 10.7°, 21.5°, 31.7°, 45.3°, and 56.6° are visible, and were attributed to the HAp phase. These peaks present diminished intensities after 7 days of immersion, indicating the degradation of the coating (supported by the mass loss presented in Figure 3
), which thus favors the growth of new bone tissue.
No discussion about other peaks was performed because all of these can be attributed to both the substrate and hydroxyapatite. Regardless of the immersion time, the XRD lines of the coatings immersed in PBS are all similar, indicating that in this case the XRD is not an adequate method for recoding the changes undergone by the coatings after immersion.
Thus, the surfaces were analyzed by the FTIR technique, which is more sensitive than XRD for confirming the new apatite phases formed after immersion in all media.
3.4. FTIR after Immersion in DMEM, SBF, and PBS
presents the FTIR spectra for HAp and HAp + SiC coatings before immersion in DMEM, SBF, or PBS. The typical bands for HAp were observed—1087, 1034, and 962 cm−1
—assigned to the presence of the P–O functional group. As it can be observed, the IR bands became wider and shifted towards lower wavenumbers after the addition of SiC into the HAp structure. Moreover, no specific absorption bands located between 770 and 680 cm−1
], corresponding to SiC stretching bonds, were revealed by FTIR analysis. Thus, it can be assumed that the addition of SiC led to some modifications in the HAp structure; some additional bands at 928 and 906 cm−1
were observed. According to the literature, these bands can be attributed to PO44−
vibrational modes, because both chemical groups share similarly spaced vibrational modes due to the similarities of PO44−
tetrahedral molecular units [81
The band intensity corresponding to 962 cm−1 in HAp was less evident for the HAp + SiC coating. This finding can be explained by the broad band located around 930 cm−1, which also includes the line from 962 cm−1.
FTIR spectra of the coated surfaces after immersion in DMEM, SBF, or PBS are presented in Figure 7
. The FTIR spectra provided lines similar to each other, which can be attributed to hydroxyapatite. Hydroxyl stretch, with its typical bands located between 3500 and 3200 cm−1
], can be observed just for the coatings immersed in SBF. These peaks are visible for the surfaces immersed for 3 and 14 days with a lower intensity and are completely invisible for the samples immersed for 1, 7, and 21 days. For the samples immersed in SBF for 3 and 7 days, a much diminished peak attributed to hydroxyl can be observed, indicating that some phosphates groups were still present on the surface.
No hydroxyl bands were seen for the coatings immersed in DMEM or PBS, regardless of the immersion time. This finding is probably due to the addition of Si into the HAp structure, which led to a reduction of hydroxyl peaks due to the extra negative charge of the silicate group, compensated by the loss of OH−
. Nakata et al. suggested that this charge is a consequence of the bonding change and symmetry of the phosphate groups which takes place due to Si addition [84
]. Carbonate bands can be usually found around 873 cm−1
) and in the region of 1650 to 1300 cm−1
The samples immersed in all solutions exhibit more or less visible carbonate bands. For the coatings immersed in SBF or PBS, these bands have a lower intensity, while for DMEM they are more intense. This finding is probably due to the competition between the phosphate and carbonate ions.
Phosphate has bands at 1190−976 (ν3
, 961 (ν1
, 469 (ν2
, and 660−520 (ν4
]. Considering our results, two weak peaks at 798 and 960 cm−1
can be found and one stronger peak at 1040 cm−1
for the coatings immersed in SBF. The intensity of the band found at 1040 cm−1
increases with the immersion time, indicating the formation of a new phosphate phase. For the coatings immersed in DMEM, a strong peak at 1031 cm−1
is observed, which is almost similar for each immersion time. For the coatings tested in PBS, the bands of phosphate appear after 3 days of immersion and grow higher after 14 days, indicating the formation of new phases. This result could be indicative of the good mineralization abilities of the coated surfaces. The FTIR spectra for the coatings immersed in PBS are noisier due to the roughness of the newly formed CaP phases.
The FTIR spectra indicate that there are some differences between the samples immersed in the investigated solutions. The most obvious change is the significant decrease of the hydroxyl peak. The hydroxyl peaks are absent in the case of the coatings immersed in DMEM and PBS. The intensity of peaks observed in the spectra of samples immersed in PBS is lower, indicating that the phase found on this surface has a low crystallinity degree.
On the other hand, the presence of the CO32−
group indicates an apatite-based structure slightly deficient in Ca [86
]. For the samples tested in PBS and DMEM, a peak located at 880 cm−1
is evident, which according to the literature can be assigned to the formation of a non-stoichiometric hydroxyapatite structure [86
]. Moreover, this peak can be also attributed to the carbonate phase. The bands ranging from 1400 to 1500 cm−1
, as well as those at 880 cm−1
, demonstrate the substitution of phosphate groups with carbonate groups in the hydroxyapatite matrix [89
3.5. In Vitro Electrochemical Performance of SiC–HAp Coatings in DMEM, SBF, and PBS
Tafel plots of the investigated coatings in DMEM, SBF, and PBS solutions are presented in Figure 8
. From the Tafel plots, the electrochemical parameters were calculated and are presented in Table 2
. It can be seen that the coatings had a better electrochemical behavior in DMEM, as compared with PBS and SBF. The coatings exhibited an almost similar evolution in PBS and SBF media.
Taking into consideration the values presented in Table 2
, it can be concluded that:
Coated samples tested in DMEM have a more electropositive value of corrosion potential (Ecorr), indicating a better electrochemical behavior, while in PBS and SBF the values are similar;
The lowest value of the corrosion current density (icorr) was recorded for coated samples tested in DMEM, followed by those in SBF and then those in PBS;
The polarization resistance (Rp) is higher for coated samples tested in DMEM, followed by those in PBS and those in SBF.
The electrochemical tests showed that the Ti6Al4V coated with SiC–HAp had a better behavior in DMEM but was affected by SBF and PBS, being less resistant in PBS. After electrochemical experiments the surface morphology and chemical composition were investigated by SEM and EDS, and the obtained images and elements distribution are presented in Figure 9
As it can be seen, the coatings were visibly degraded in PBS when compared to DMEM and SBF. Nevertheless, the sample in PBS media registered the lowest Ca/P ratio with a value of 1.27 when compared to that in SBF media (ratio of 1.43) and DMEM (ratio of 1.72). SEM images also revealed that PBS favors the appearance and propagation of cracks in the HAp + SiC coating, a phenomenon that was not visible for the coatings immersed in SBF and DMEM.