The phase structure of the MAO coating was investigated by powder XRD analyses as shown in Fig. 1. As seen in XRD spectra, the phases of Zr, cubic-ZrO2, meta-stable Ca0.15Zr0.85O1.85 and Ca3(PO4)2 were detected on the MAO surface. Cubic-ZrO2 and Ca3(PO4)2 were observed as major phases, while Zr was found as minor phases in the coating structure. The signal of Zr on XRD spectra derives from the substrate and metallic compounds on the coating. Firstly, the ZrO2 was formed by the reaction of oppositely charged Zr4+ and OH− ions under high pressure and high temperature on the micro discharge channels at the initial steps of MAO. The instant localized temperature in micro discharge channels reached up to 2500 K through the MAO process as reported in the literature 44. Therefore, stable cubic-ZrO2 was observed through whole surface. Moreover, the phase of ZrO2, which serve as nucleation sites, contributed to the formation of Ca-based phases such as Ca3(PO4)2, Ca0.15Zr0.85O1.85 and Ca10(PO4)6(OH)2 41,42. Positively charged Ca2+ and negatively charged PO43− ions derived from electrolyte reacted with each other on ZrO2–based micro discharge channels. And then, Ca3(PO4)2 was formed on the MAO surface. Simultaneously, the Zr4+ from the substrate and the Ca2+ and OH− from electrolyte combined with each other on micro discharge channels. Then, they form meta-stable Ca0.15Zr0.85O1.85 45.
The FT-IR spectra of the chitosan-based MAO coatings was given in Fig. 2. It designated the characteristic bands of chitosan, ZrO2 and calcium apatite-based structures. The FTIR peaks located at 560–570, 645–655, 1028, 1089, 1150, 1425, 1590, 1657, 2140–2165, 2340–2380, 2872, 3360–3370 and 3730–3750 cm− 1 correspond to PO43−, OH-, PO43−, C-O-C, C-N, N-H, N-H, -NH2, CO, P-H, C-H, O-H and OH−, respectively 37,45−53. Two peaks located at 1089 and 1150 cm− 1 are characteristic absorption peak of C-O-C and C-N stretching vibration mode, respectively 37,51. The absorption band peaks at 1425 and 1590 cm− 1 correspond to N-H band 37,50,51. The stretching vibration band peak at 1657 cm− 1 corresponds to –NH2 37. The stretching vibration band peaks at 2872 cm− 1 attributes to C-H in methyl or methenyl 37. The approximately stretching vibration of non-associated peaks at 3360–3370 cm− 1 correspond to O-H band 52. All of these peaks verify the existence of chitosan-based layer structure on the MAO surface 37. Furthermore, the other peaks support the presence of c-ZrO2, Ca3(PO4)2 and apatite. The characteristic band peak at 1028 cm− 1 verify to the existence of Ca3(PO4)2 53. The absorption band peak at 2140–2165 cm− 1 verify to the existence of c-ZrO2 45. The stretching vibration, libration-deformation, stretching vibration and stretching vibration band peaks at 560–570, 645–655, 2340–2380 and 3730–3750 cm− 1 verify to the existence of apatite 45–49. However, crystalline apatite was not observed on the MAO surfaces by XRD as seen in Fig. 1. Thus, it could be concluded that the MAO coatings contained an amorphous apatite structure because it could not kinetically be transformed to crystalline form during MAO process.
The surface morphologies of the MAO and chitosan-based MAO coatings were investigated by SEM as seen in Fig. 3. The surface of the MAO coatings was very porous and rough owing to the presence of micro sparks during the MAO process. Many micropores and voids were found on the MAO surface. The cracks were found on the MAO surface because the thermal stresses appeared between the localized hot surface and cold electrolyte during the process. It is well known that these types of porous and rough bioceramic surfaces are beneficial for cell attachment, proliferation and tissue growth under body conditions for biomedical implant applications. All pores and voids were filled with antibacterial type of chitosan polymer structure after the MAO surface was coated by dip coating method. And then, homogeneous antibacterial chitosan-based MAO surfaces were fabricated on zirconium. After being coated with a chitosan layer on the MAO surface, any micropores, voids and thermal cracks were observed as shown in Fig. 3b. The spherical chitosan structures were monitored on the surface as expected. Thus, it suggests that chitosan-coated MAO surfaces were completely covered.
The elemental distribution found on both surfaces were analyzed by EDX-mapping as illustrated in Fig. 4. The elemental amounts of both surfaces were given in Table 1. As expected, only Ca, P, O and Zr elements were detected on the MAO surface. The Ca, P and O elements originated in calcium acetate and calcium glycerophosphate-based electrolyte as Zr came from the metallic substrate as expected. Furthermore, all detected elements homogenously distributed through whole the MAO surface were shown in Fig. 4a. Besides the existence of Ca, P and O elements, C was detected on the chitosan-based MAO surface. This element was uniformly dispersed during the surface at post-coating chitosan layer. The chitosan structures naturally contained the C and O elements. However, no Zr elements were observed on the chitosan-based MAO surface. It is concluded that Zr-based oxide structures were found inner layer and the outer surface mainly consisting of Ca-based bioactive and biocompatible elements and phase structures. This situation clearly supported the contribution of ZrO2 on the formation of Ca-based structures.
Table 1
EDS spectra results of the MAO and chitosan-based MAO coatings
Elements
|
MAO coating
|
Chitosan-based MAO coating
|
Wt. %
|
At. %
|
Wt. %
|
At. %
|
Zr
|
23.72
|
6.81
|
-
|
-
|
O
|
42.14
|
69.04
|
49.03
|
46.36
|
Ca
|
24.68
|
16.14
|
8.09
|
3.05
|
P
|
9.46
|
8.00
|
4.29
|
2.09
|
C
|
-
|
-
|
38.45
|
48.42
|
The wettability of both surfaces was investigated by a sessile drop - contact angle measurement technique as shown in Fig. 5. The average contact angle values of the MAO and chitosan-based MAO surfaces were measured as 94.0° ± 0.3 and 113.5° ± 0.2, respectively. All measurements were repeated for three times to get an average value of the wettability of the surfaces. Both surfaces had hydrophobic properties since the average contact angle values were bigger than 90°. However, in terms of compassion, the chitosan-based MAO surface indicated hydrophobic character with respect to the MAO surface. The wettability mainly depends on morphological structures/chemical compositions of the surfaces. The MAO surfaces which had many voids and thermal cracks were porous structure as observed in Fig. 3a. The MAO surfaces usually exhibit hydrophilic properties owing to the capillary effect on the liquid of pores 54. Thus, the water molecules droplet on the MAO surfaces was easily absorbed and spread compared to homogenous chitosan-based biopolymer surface. The highest initial contact angle value was in agreement for the chitosan-coated substrate as reported in the literature and this can be attributed to the basis of its chemical properties 55. Observed large initial contact angle value can indicate the reorganization of the molecule which is presumably associated with the methyl moieties of the residual acetyl groups along the polysaccharide backbone 56. Therefore, the wettability of chitosan-based MAO surface was lower than the one of the MAO surface.
In vitro immersion test of the MAO and chitosan-based MAO surfaces were carried out under 36.5 °C in SBF for 14 days. It is well known that this test gives an information about predicting bioactivity of both surfaces. At-post immersion in SBF, the phase structure, surface morphology and elemental distribution of both surfaces were analyzed by XRD (Fig. 6), SEM (Fig. 7) and EDX-mapping (Fig. 8), respectively. Moreover, the amount of the elements formed on both surfaces at post-immersion in SBF was given in Table 2. As seen in Fig. 6, a TCP (Ca3(PO4)2) and hydroxyapatite (Ca5(PO4)3(OH))structure was detected as major phase on both surfaces. The Ca2+ ions, which released from proteins, adsorb PO43− ions by electrostatic interactions in SBF solution 57. Simultaneously, they react with each other and form Ca3(PO4)2 at early stages of immersion in SBF. And then, they react with OH− ions and transform to Ca5(PO4)3(OH) through the immersion process. The formation mechanism of hydroxyapatite structure occurred on different types such as un-doped and an antibacterial Ag, Cu and Zn-doped MAO surfaces at post-immersion in SBF were discussed in detail in our previous studies 38−42. The SBF immersion test revealed that chitosan layer was favorable for hydroxyapatite formation. The bioactivity of chitosan was originated due to a large number of protonated amino groups on chitosan surface. Chitosan’ surface can absorb OH− ions in SBF via hydrogen bond and electrostatic attraction. Eventually, they would be adsorbed the Ca2+ and PO43− in solution by electrostatic attraction. Finally, the reaction of them under SBF conditions form the bone-like apatite on chitosan-based MAO surface 21. Furthermore, the chitosan layer contribute to nucleate hydroxyapatite because it contains a large amount of OH 58. It was observed that the amount of crystalline apatite structure formed on chitosan-based MAO surface was greater than the one on the MAO surface as shown in Fig. 6. The original porous bioceramic MAO and spherical biopolymeric chitosan-based MAO surfaces were filled with a new layer at post immersion in SBF. As shown in Fig. 7, a new apatite layer was completely deposited on both surface layer, whereas the chitosan-based MAO surface was nonporous and had polymer structure with respect to the MAO surface. The Ca, P and O are necessary basic elements for the formation of apatite. Only, Ca, P and O elements on both surfaces were observed at post immersion in SBF. Furthermore, all of these were uniformly distributed through the whole surface as shown in Fig. 8. Furthermore, the elemental amount of both surfaces at post-immersion in SBF are the similar as given in Table 2. However, it is clear that a new apatite layer on the chitosan-based MAO surface seem as crack-free and homogenous than the MAO surface. Therefore, it is stated that a chitosan layer on the MAO surface contributed on the formation of apatite structure and improved the bioactivity.
Table 2
EDS spectra results of the MAO and chitosan-based MAO coatings at post-immersion in SBF
Elements
|
MAO coating
|
Chitosan-based MAO coating
|
Wt. %
|
At. %
|
Wt. %
|
At. %
|
Ca
|
33.23
|
18.62
|
33.32
|
18.69
|
P
|
18.19
|
13.19
|
18.18
|
13.19
|
O
|
48.57
|
68.19
|
48.49
|
68.12
|
The antibacterial activities of MAO and chitosan-coated MAO surfaces were examined by agar diffusion test and the results are given in Fig. 9a-9c. The minimum inhibition zone for both strains was obtained with the MAO surfaces. The MAO surface exhibited 5.5 ± 0.7 and 4.2 ± 0.3 mm inhibition zones against E. coli and S. aureus, respectively. It was observed that after the chitosan coating of the surface, the inhibition zones obtained against bacteria increased significantly. Chitosan-based MAO surfaces exhibited 21.6 ± 1.3 and 13.7 ± 0.9 mm inhibition zones against E. coli and S. aureus, respectively. It was determined that chitosan-based MAO surfaces have 74.5% more antibacterial activity against E. coli than the MAO surfaces. For S.aureus, chitosan-based MAO surfaces exhibited 69.3% more antibacterial activity than the MAO counterparts. This result can be related to the antibacterial properties of chitosan coating. Chitosan is the deacetylation product of the chitin molecule. Chitin is a linear biopolymer formed by the bonding of N-acetyl D-glucosamine units by glyosidic bonds 59. Chitin is insoluble in many solvents due to the compact structure. The lack of solubility in dilute acid or alkaline solvents, especially in water, limits the chitin usability 60. In order to increase its solubility and usability, the chitin is subjected to de-acetylation with NaOH and high solubility chitosan is formed. Chitosan is a straight-chain polymer consisting of D-glucosamine and N-acetyl D-glucosamine 61. It contains more amine groups and is easily soluble in acidic solutions. Chitosan, which is physically, chemically and biologically compatible, is known to have medical activities such as antidiabetic, antimicrobial, antioxidant and antitumor 62. The antibacterial activity of chitosan is due to its polycationic structure 63. Positively charged chitosan interacts with the negatively charged components of the bacterial cell causing disruptions in normal cell metabolism 64. It is reported in the literature that many materials coated with chitosan exhibit different levels of antibacterial properties. Zhang et al. reported that chitosan-TiO2 composite materials exhibit strong antimicrobial activity against E. coli, S. aureus, C. albicans and A. niger 65. In another study, Munteanu et al. found that chitosan-coated polyethylene surfaces provided a 100% inhibition against S. enteritidis after 48 hours of interaction, while providing 96.43% inhibition against E. coli 66.
Another important result obtained from the antibacterial test is that chitosan-coated MAO surfaces show higher effect against E. coli compared to S. aureus. This result shows that in general, the chitosan-coated MAO surface is more effective against gram negative compared to gram positive. It was determined that the antibacterial effectiveness of chitosan-coated MAO surfaces against E. coli is 1.58 times more than S. aureus. This result can be explained by the differences in the cellular structure of gram positive and gram negative bacteria. The fact that the gram-negative bacteria surface has more hydrophilic character compared to gram-positive bacteria makes them more susceptible to chitosan 67. The high hydrophilic property leads to greater interaction with chitosan and large changes in the structure and permeability of the cell membrane. These alterations result in bactericidal effects and bacterial death 68. Similar studies have demonstrated that chitosan-coated surfaces have a higher inhibitory effect against gram-negative bacteria. Munteanu et al. (2014) examined the inhibitory effect of chitosan-coated films with two Gram-negative bacteria, namely S. enteritidis and E. coli, and a Gram-positive bacteria, L. monocytogenes, and reported high inhibition in gram negatives. Esmaeili et al. reported that chitosan-coated nanoparticles exhibited significant antibacterial effect against gram negative bacteria 69. As a result, it was determined that chitosan-based MAO surfaces have high antibacterial properties compared to the MAO surfaces and exhibit a broad spectrum activity by affecting both gram negative and gram positive bacteria.