**4. Discussion**

The amounts of the released nickel and chromium ions are di fferent, which, among other reasons, results from the content of these elements in the alloy (about twice as much nickel as chromium). However, the ratio of the released nickel ions to the chromium ions is about 3 to 4 times higher. So, one should presume that the examined coatings constitute a better barrier for chromium than for the nickel. This can result from the di fference of the size of the nickel Ni2<sup>+</sup> and chromium Cr3+ ions, which equals about 20%.

The performed tests showed that, in the case of the Ti(C, N)-type coating, the amounts of Ni and Cr ions released into each of the tested environments was much lower than in the case of the alloy without a coating. In the case of each environment, the statistical analysis of the obtained results demonstrated a statistically significant decrease in the amount of released ions in the coated samples in respect of the samples without a coating. No statistically significant di fferences in the amount of released ions were observed between the samples covered with the particular coatings. The lack of significant di fferences in the penetration of the ions should not be surprising; the coatings have a similar construction and corrosion resistance. However, a more thorough analysis of the results makes it possible to notice a certain tendency showing that, in most cases, the amount of ions penetrating the coatings from groups S2, S3, and S4 is a little higher than that of the ions penetrating the coatings from groups S1 and S5, and this di fference results from their thickness, as coatings S2 and S3 are the thickest and so, they should constitute the best barrier. This can be explained in two ways. First, these coatings are more fine-grained. Their fine-grained structure can be demonstrated by the results of di ffraction tests included in the study [31] While the results presented there refer to the phase identification of coatings, as well as their texture and stresses, in the analysis of the width of the reflexes from phases Ti(C, N) (samples S2, S3, S4), we observe their slight widening in respect of phases TiC (S1) and TiN

(S5), which can prove their more fine-grained structure. A fine-grained structure significantly increases the amount of grain boundaries, and these, as we know, are the areas of facilitated di ffusion. So, nickel and chromium from the alloy can penetrate the coating more easily. Also, one should take into account the fact that these processes take place at ambient temperatures, where other di ffusion mechanisms are hindered. Another reason for such a behaviour of the coatings can be their crystallographic structure. Carbide TiC has a similar structure of the crystal lattice as that of nitride TiN, and they di ffer only in the parameters of the lattice. In the carbonitrides being formed, the carbon atoms are successively replaced by the nitrogen atoms (the latter are placed in the position of the former, without a change in the crystal lattice). As carbon atoms are bigger than nitrogen atoms, this change causes a certain relaxation of the lattice, which can create a situation of facilitated di ffusion.

In the analysis of the amount of ions released into the particular examined solutions, no statistically significant di fferences were established, ye<sup>t</sup> certain tendencies could be observed. The highest amount of ions released into the environment of a 0.9% NaCl solution results from the aggressiveness of the chlorine ions. Chloride ions have a clear electronegative character and they favour the metals' passing from the atomic state into the ionic state. This is especially visible in the alloy without a coating. The amounts of the ions released from the pure metal are slightly higher, both in the environment of the 0.9% NaCl solution and artificial saliva, as both solutions contain chlorine ions. The slightly lower amount of ions released into distilled water can be explained by the di ffusion into a non-saturated environment. The lowest amount of ions from the coated samples were released into the solution of artificial saliva, which might be explained by the fact that the latter contains phosphates and sulfides, which can form low-soluble phosphates and sulfides with the substrate metals, and they, in turn, can block the discontinuity of the structure and the porosity of the coating, thus hindering the passing of the ions.

Covering metal alloy elements with coatings can significantly reduce the cytotoxicity of these elements, thus providing the possibility of their use in contact with various tissues of the human body. The investigations performed at many scientific centres have confirmed the positive results of the use of di fferent coatings. In a study concerning the application of diamond like carbon (DLC) coatings on metal elements, the authors demonstrated this in short-term and long-term investigations in vivo performed on rat males, breed WISTAR. The modified samples were implanted under the skin for 24 h, 1, 4 and 26 weeks. After that time, the animals were put down and the tissues surrounding the modified elements and the spleen were collected. The evaluation of two routine dyed pieces of tissue and spleen showed that: 1) the carbon coating protects the tissue surrounding the metal implants against metal ion penetration, 2) no immunological reaction of the tissue or the spleen occurs, whereas the non-coated elements cause a strong reaction, 3) the statistically important di fferences (pb0.05) refer to the number and type of the cell elements and the degree of reaction in respect of the modified and non-modified elements [40]. Also, studies of similar coatings were performed, where the obtained amounts of released metal ions were correlated with the estimated biological response of the cell viability test (osteoblast cell line Saos-2) and the bacterial colonization test (strain Escherichia coli DH5 α). The results showed that depositing hybrid DLC-type coatings by means of the magnetron sputtering radio frequency plasma assisted chemical vapour deposition (RF PACVD/MS) method makes it possible to obtain an air-tight coating, which prevents di ffusion of harmful elements from the metallic substrate [41].

A reduced toxicity level is also exhibited by nitride and carbide coatings. Jang et al. studied biological Ti samples covered with TiN and 3-1-2,5-diphenyl tetrazolium bromide test (MTT) coatings, among other things, by examining their vitality by means of the MTT test after 8 days of being placed in the medium. The test showed a higher survivability of the cells from the group coated with TiN (by 125%) and TiAlN (by 117%) compared to the pure Ti group (100%) [42]. In the studies of Bramm et al., titanium was coated with titanium carbide TiC in the pulsed laser deposition (PLD) technology and evaluated in vitro in 3 cell lines as well as in vivo in respect of the integration with the bone, in tests performed on animals. The homogeneous TiC coatings covering titanium had a positive e ffect on the

bones forming the cells, both in vitro and in vivo. According to the authors, these e ffects result from many factors, including both the chemistry of the coating and its morphology, with the consideration of the proper roughness. The TiC coatings simultaneously increase the implant's hardness and protect titanium from the aggressive attacks of the body fluids and tissues. The authors conclude that the use of TiC coatings on titanium improves the biocompatibility; also, with the appropriate roughness, it stimulates the proliferation of osteoblasts, their adhesion and diversity, as well as improves the osteointegration of the implant [43].

The biological tests performed on Ni–Cr alloys covered with the presented coatings also showed an improvement of the biocompatibility of the coated alloy in reference to the non-coated alloy. The evaluation by means of the MTT viability test performed on human microvascular endothelial cells coming from the skin—HMEC-1—showed that, in the case of the samples incubated for 24 and 96 h, we can observe statistically significant di fferences (with the value *p* < 0.05) between the non-coated samples and all the other samples coated with Ti (C, N) [36]. Also, the investigations with the use of fibroblasts proved that no Ti(C, N) coating a ffects the activity of the oxidoreductive enzymes, ether in the case of the direct cell culture or the application of extracts of the tested materials [37]. Such a behaviour of the Ti(C, N)-type coatings has also been observed by other researchers. Serro et al. [44] compared the cytotoxicity of 48-h extracts from implants coated with carbonitride Ti(C, N) (containing 21.6% C) and nitride TiN on mouse fibroblasts. Examining the morphology and biological activity and comparing them with the cells bred in a normal medium, they did not observe a cytotoxic e ffect of Ti (C, N) and TiN coatings on those cells. In the studies concerning a TiN coating on a nickel alloy [45], it has been established that it does not cause a cytotoxic irritation; a significant increase of the gum fibroblast on the surfaces of nickel alloys coated with TiN has been recorded, as opposed to the surface of the alloys without a TiN coating.

It can be inferred from the performed studies that there are no significant di fferences in the amounts of the ions which have passed through the particular coatings. Also, their biological properties are similar [36,37]. So, it is impossible to point to the best one. During clinical proceedings, their properties should rather be considered with regard to a particular application. As it was demonstrated in earlier studies, they exhibit big di fferences in hardness, modulus of elasticity and shear resistance [34].

The positive e ffect of the coatings is caused by various factors. Undoubtedly, a decrease in the harmful ions has basic importance. Ti(C, N)-type coatings additionally prevent oxidation, which, as is suggested by Toshifumi, could have a significant e ffect on the surface wettability [46]. This parameter is important, e.g., during the cells' adhesion to the substrate. The wettability measurements of the presented coatings demonstrated their higher hydrophilicity than alloy Ni–Cr [34]. Also, Li confirmed that NiTi coated with TiN exhibits a much higher hydrophilicity than in the case when no coating is applied. Additionally, he pointed to a significant role of the surface roughness [47], which can be especially important during the osteointegration of the implants with the bone [48].

Correlating the results of the amounts of the ions released from alloys with biological responses, we can see that the presence of the coatings causing a reduction of the amount of the ions released into the environment clearly favours a reduction of the alloys' cytotoxicity.
