Our aim was to determine both the applicability of the PPAAm layer on these crystalline zirconia ceramics and also the minimal possible film thickness that would allow cells to spread optimally. We carried out the standard plasma deposition process in dependence on the treatment time and described, among other things, the interaction of MG-63 cells with the PPAAm film deposited in different thicknesses on the Y-TZP ceramic.
3.1. Physico-Chemical Surface Properties of PPAAm-Treated Y-TZP Ceramic
As a precondition for our further investigations, we studied the crystallographic structure of the ceramic substrate by X-ray diffraction analysis before and after plasma treatment to determine the influence of the plasma process.
The GIXRD analysis of the untreated and PPAAm-treated ceramic samples confirmed clearly, in agreement with the data file JCPDS 42-1164, the tetragonal crystal modification of Y-TZP (Figure 4
). The undesired monoclinic phase was not detectable. The MW plasma process for the deposition of PPAAm has no influence on the tetragonal crystal structure of Y-TZP present.
The chemical composition and molecular structure of PPAAm thin films on ceramic (not shown) could be confirmed by means of FT-IRRAS. The FT-IRRAS spectra of PPAAm showed a high retention of the structural properties of the monomer allylamine H2
for the MW plasma deposition method used here in accordance with [13
A further approach in our investigations was the gradual reduction of the PPAAm film thickness. We searched for the minimal possible film thickness, which would allow cells to behave optimally. For all samples (Si-wafer), one and the same standard plasma process parameter was used, only the gross treatment times varied (see Section 2.2
) and, therefore, the film thickness. A quasi-linear film growth could be found from 15 s until about 480 s (Figure 5
). At 960 s, etching processes more frequently accompany the deposition process, followed by a greater degree of cross-linking of the plasma polymer, reduced film thickness, a loss of surface functional groups, and increasing WCA.
Layer formation and growth of the plasma polymer film PPAAm are of particular interest and can be explained by the results of Michelmore et al. [9
] and Akhavan et al. [23
]. They shed some light on the growth mechanisms of the very first layers of plasma polymer films, also for PPAAm. Michelmore et al. [9
] verified a very smooth PPAAm film growth, and a continuous layer formation even for very thin films (<2 nm) from the very earliest stages on a silicon surface. The double bond in the precursor allylamine encourages a better cross-linking in PPAAm and seems to contribute to the smooth layer formation. Island-like structures could be excluded for PPAAm. These results confirm our observations of very smooth PPAAm films (480 s: Rq = 1.9 ± 0.2 nm; 960 s: Rq = 3.8 ± 0.9 nm). Akhavan et al. were, again, able to demonstrate that the formation of metallic (zirconium/titanium-) carbide and oxycarbide bonds in the initial stages of the film formation led to the excellent film adhesion of the plasma polymer. A 2D-like, layer-by-layer (Frank-van der Merve mechanism) manner of growth was observed [23
Plasma-treated surfaces are very reactive upon completion of the plasma deposition process. So-called post plasma processes, especially oxidation and hydrolysis, take place after storage in ambient air, influencing the wettability and surface energy by changing the surface chemical compositions. The wettability is determined by the charge and polarity of the surface functional groups. Therefore, the water contact angle (WCA) measurements were always carried out 30–60 min after the plasma process in each case, to get comparable results.
shows the WCA of untreated (time point 0 s) and PPAAm-coated smooth and rough ceramic surfaces as a function of the plasma treatment time. In the case of the smooth ceramic (Rq ~273 ± 40 nm), the WCA are in a range between 50° and 60° (54.9 ± 4.4°), whereas the WCA values of the rough ceramic (Rq~1320 ± 20 nm) vary more strongly between 40° and 60° (46.5 ± 12°) and are more hydrophilic than the smooth ceramic surfaces. For both roughnesses, the WCA are in the hydrophilic region in which the wettability is optimal for cell responses [24
]. Even a very short plasma treatment time of 15 s is sufficient to shift the WCA of the untreated samples into the hydrophilic region, here especially apparent for the rough surfaces.
The high WCA values of the untreated rough ceramic may be contributed to the enlargement of the effective surfaces area combined with higher hydrocarbon content. Our recent experiments by Rebl et al. [15
] using polished and machined Ti showed similar tendency concerning WCA and roughness: For Ti-P (Ra 0.045 µm) and Ti-M (Ra 0.315 µm), the WCA values results in ca. 60° vs. 80°, respectively.
The zeta potential measured at pH 7 for the untreated rough zircon oxide ceramic is strongly negative at −53.1 ± 1.4 mV. The PPAAm nanofilm (480 s; on Si-Ti wafer) led to a positively charged surface (pH 6) at +32.8 ± 12 mV immediately after the deposition process and +35.5 ± 2 mV after 180 days storage in ambient air. The trend is consistent with our results in [13
The surface energy and water contact angle of untreated and PPAAm-coated (120 and 960 s) smooth ceramic surfaces demonstrated that the PPAAm treatment led to an enhancement of the total surface energy, including their polar and disperse parts (Figure 7
A) and to decreasing WCA (Figure 7
B). The total surface energy of the untreated smooth ceramic increased from about 37 ± 2 mN/m to about 50 ± 0.5 mN/m for both PPAAm coated surfaces (for 120 s and 960 s brutto, respectively). Also, the disperse and polar part of the surface energy increased on PPAAm. The disperse part of the untreated ceramic 24 ± 3 mN/m rose to 30 and 33 mN/m and the polar part of the untreated ceramic 14 ± 5 mN/m to 20 and 16 mN/m for 120 s and 960 s PPAAm. Furthermore, the shorter PPAAm treatment time of 120 s has a higher polar and lower disperse part than 960 s PPAAm. Possible reasons for this may be a lower influence of the etching processes or more surface functional groups through a lesser degree of cross-linking.
The XPS surface composition, as a function of the plasma treatment time (Figure 8
) for smooth and rough ceramic surfaces shows that, with increasing treatment time, the ceramic surface will be covered systematically by the plasma polymer film PPAAm. The elements of the ceramic substrate Zr, Y, in slight traces Al, Si, Na, and also O disappeared; meanwhile, the components of PPAAm C and N increased. A complete covering by PPAAm is reached at about 200 s. The significantly higher roughness of the rough ceramic led to higher standard deviations in C, O, and N. Zr and Y are in the same dimensions as is the case for smooth ceramic surfaces.
The bar diagram (Figure 9
) illustrates the surface composition of selected ceramic samples that were used in cell culture. The N/C ratios of PPAAm coatings for as little as 15 s is at 20% (not shown here) and increases continuously: at 60 s to 26%, 120 s to 26.4%, 480 s to 25.8%, and 960 s to 26.8%, whereby the theoretical N/C value of the precursor allylamine is 33%. The PPAAm films formed until about 120 s of treatment time already contain a considerable number of nitrogen functional groups at the surface.
Y-TZP ceramic surfaces that are negatively charged can be modified by the plasma polymer PPAAm under retention of the tetragonal crystal structure. Very smooth, positively charged PPAAm films could be deposited. In accordance with Michelmore et al. [9
] and Akhavan et al. [23
], a 2D-like, layer-by-layer (Frank-van der Merve mechanism) manner of growth can be assumed from the very earliest stages. Even short plasma treatment times led to PPAAm films with relatively high N/C ratios of about 20–26%. The WCA are optimal, lying in the hydrophilic range, and the total surface energy including their polar and disperse parts are enhanced on PPAAm. Thinner PPAAm films (<10 nm) seem to be beneficial in every respect and showed optimal conditions for adhesion and spreading of osteoblasts.
For future dental applications it is essential to evaluate the long-term stability of the PPAAm nanolayer on these Yttria-stabilized, tetragonal zirconia polycrystal ceramics (at least one year) as well as the in vivo effectiveness in the jawbone.
3.2. Cell Behavior on Titanium (Ti) and Ceramic Surfaces Bioactivated with Plasma Polymer (PPAAm)
The plasma nanolayer PPAAm is convincing in its cell attractiveness. The 480 s plasma process on Ti, which results in a homogeneous nanolayer, allowed the cells to spread significantly in comparison with the uncoated Ti control, which we had already observed earlier [20
]. In this work, it was of interest to determine whether a reduced plasma process time on Ti could induce the same spreading behavior. The PPAAm plasma treatment time of 60 s and 480 s gross, which corresponds to a layer thickness of about <10 nm and ~30 nm resp., revealed for both N/Cs ratios of about 26%. However, the cell-attractive characteristic of this nanolayer was obvious (Figure 10
). After 1 h, cells on Ti PPAAm spread well and reached values of 1085.4 ± 55.5 µm2
and 1365.6 ± 68.7 µm2
for the PPAAm processes 60 s and 480 s, respectively (control: 713.8 ± 51.0 µm2
). The actin cytoskeleton is already more pronounced in stress fibers after a 60 s deposition time of plasma polymerized allylamine.
Analogous to our investigations of PPAAm coatings on different materials (see Table 1
), we prepared and analyzed the effects of conditioning ceramic surfaces with PPAAm, especially with regard to their further use as dental materials. The well-spread morphology also on PPAAm-coated ceramic surfaces is obvious (Figure 11
). Ceramic on its own provides the cells with a surface characteristic, which leads to cell spread. In addition, the coating allows the cells to melt into the ceramic surface structure. The cells mimic the topography and the underlying structure appears through the flattened cell. These phenomena could also be observed with cells on metal surfaces, especially on corundum-blasted sharp-edged Ti coated with PPAAm, process duration 960 s gross resulting in a 50 nm layer [15
]; or on plasma chemical oxidized surfaces additionally coated with PPAAm [11
]. On PPAAm-modified ceramic, the cells are able to adhere immediately in the first 10 min and to a great degree of more than 80% compared with the untreated ceramic and the TCPS control at 81.4 ± 9.9%, 19.3 ± 2.9%, and 30.6 ± 4.7%, respectively (Figure 12
Summarizing all results with ceramic surfaces as well as other biomaterials tested (see Table 1
), we can say that the moderately positively charged plasma polymerized allylamine (PPAAm) nanolayer is very cell attractive. This phenomenon can be seen in many cell biological concerns: (i) On PPAAm-modified materials, the cell spreading is very fast, cells are flattened, and cells melt into the surface structure [15
], and (ii) this cell attractiveness leads to the next phenomenon that cells “forget” the underlying surface structure. This could be observed for the first time by our group. Normally, on material topographies, cells feel the underlying topography, i.e., cells are aligned on micro-grooves or machined Ti. But if these materials additionally PPAAm-modified, the cells not only adhere better and spread faster, but also override the topography [10