Human osteoblast and fibroblast response to oral implant biomaterials functionalized with non-thermal oxygen plasma

Abstract

Plasma-treatment of oral implant biomaterials prior to clinical insertion is envisaged as a potential surface modification method for enhanced implant healing. To investigate a putative effect of plasma-functionalized implant biomaterials on oral tissue cells, this investigation examined the response of alveolar bone osteoblasts and gingival fibroblasts to clinically established zirconia- and titanium-based implant surfaces for bone and soft tissue integration. The biomaterials were either functionalized with oxygen-plasma in a plasma-cleaner or left untreated as controls, and were characterized in terms of topography and wettability. For the biological evaluation, the cell adhesion, morphogenesis, metabolic activity and proliferation were examined, since these parameters are closely interconnected during cell-biomaterial interaction. The results revealed that plasma-functionalization increased implant surface wettability. The magnitude of this effect thereby depended on surface topography parameters and initial wettability of the biomaterials. Concerning the cell response, plasma-functionalization of smooth surfaces affected initial fibroblast morphogenesis, whereas osteoblast morphology on rough surfaces was mainly influenced by topography. The plasma- and topography-induced differential cell morphologies were however not strong enough to trigger a change in proliferation behaviour. Hence, the results indicate that oxygen plasma-functionalization represents a possible cytocompatible implant surface modification method which can be applied for tailoring implant surface wettability.

Figure 1

SEM images of ATZ (a), Y-TZP (b) and titanium (c) surfaces for osteoblast culture. Magnifications were set to 1000x (first row) and 5000x (second row). Arrows point to macroscopic grooves and gaps on ceramic surfaces.

Figure 2

Three-dimensional topographical characterization of osteoblast surfaces by interferometry. Surface parameters describing the topography of biomaterial surfaces were (a) S_a (average surface height deviation amplitude), (b) S_q (root-mean-square deviation), (c) S_z (ten-point height of surface topography), (d) S_sk (skewness), (e) S_dr (surface enlargement compared to a totally flat reference area), (f) S_dq (root mean square gradient, existence of surface slopes), (g) S_tr (texture aspect ratio), (h) S_pd (density of peaks) and (i) S_pc (curvature of peaks). Plots show mean values (n = 4) ± SEM. Statistically significant differences (p < 0.05, Tukey´s HSD test) were marked with brackets above the corresponding bars.

Figure 3

SEM images of ATZ (a), Y-TZP (b) and titanium (c) surfaces for fibroblast culture. Magnifications were set to 1000x (first row) and 5000x (second row). Arrows point to surface depressions on ceramic surfaces.

Figure 4

Three-dimensional topographical characterization of fibroblast surfaces by interferometry. Surface parameters describing the topography of biomaterial surfaces were (a) S_a (average surface height deviation amplitude), (b) S_q (root-mean-square deviation), (c) S_z (ten-point height of surface topography), (d) S_sk (skewness), (e) S_dr (surface enlargement compared to a totally flat reference area), (f) S_dq (root mean square gradient, existence of surface slopes), (g) S_tr (texture aspect ratio), (h) S_pd (density of peaks) and (i) S_pc (curvature of peaks). Plots show mean values (n = 4) ± SEM. Statistically significant differences (p < 0.05, Tukey´s HSD test) were marked with brackets above the corresponding bars.

Figure 5

Characterization of biomaterial surface wettability by contact angle measurement. (a) Biomaterials intended for osteoblast cultures. (b) Biomaterials intended for fibroblast cultures. Graphs show mean values (n = 8) ± SEM. Statistically significant differences (p < 0.05, Tukey´s HSD test) were marked with brackets above the corresponding bars.

Figure 6

Immunofluorescence of phalloidin-labelled actin (red fluorescence) in osteoblasts cultivated for 1, 3 or 7 days on plasma-functionalized and control surfaces.

Figure 7

Indirect immunofluorescence of the phalloidin-labelled actin (red fluorescence) and the focal adhesion protein vinculin (green fluorescence) in osteoblasts cultivated for 1, 3 or 7 days on plasma-functionalized and control surfaces. Nuclei were counter-stained with Hoechst 33342 (blue fluorescence). Arrows point to actin stress fibres.

Figure 8

Quantitative morphometric analysis of osteoblast morphology after 1 and 3 days of culture on untreated and plasma-functionalized surfaces. Since the cells had reached confluence at day 7, no morphometric analysis was possible at day 7. Data are presented as mean values (194 < n < 373) ± SEM. Statistically significant differences (p < 0.05, Dunn´s HSD test) were marked with brackets above the corresponding bars. “*” marks statistically significant differences between day 1 and 3.

Figure 9

Immunofluorescence of phalloidin-labelled actin (red fluorescence) in fibroblasts cultivated for 1, 3 or 7 days on plasma-functionalized and control surfaces.

Figure 10

Indirect immunofluorescence of the phalloidin-labelled actin (red fluorescence) and the focal adhesion protein vinculin (green fluorescence) in fibroblasts cultivated for 1, 3 or 7 days on plasma-functionalized (large images) and control surfaces (inserts). Nuclei were counter-stained with Hoechst 33342 (blue fluorescence).

Figure 11

Quantitative morphometric analysis of fibroblast morphology after 1 and 3 days of culture on functionalized and control surfaces. Since the cells had reached confluence at day 7, no morphometric analysis was possible at day 7. Data are presented as mean values (145 < n < 300) ± SEM. Statistically significant differences (p < 0.05, Dunn´s HSD test) were marked with brackets above the corresponding bars. “*” marks statistically significant differences between day 1 and 3.

Figure 12

Metabolic activity (a) and total cell number per area determined by counting the cells on micrographs (b). The percentage of alamarBlue reduction in the supernatant refers to a 100% reduced control. Data are presented as mean values ± SEM (data collected on 6 biomaterial discs). No statistically significant differences were found between the different biomaterial groups. “+” marks statistically significant differences between day 3 and day 7 and “#” between day 1 and day 7.

Figure 13

Metabolic activity (a) and total cell number per area determined by counting the cells on micrographs (b). The percentage of alamarBlue reduction in the supernatant refers to a 100% reduced control. Data are presented as mean values ± SEM (data collected on 6 biomaterial discs). Statistically significant differences between the different biomaterial groups were marked with brackets above the corresponding bars. “*” marks statistically significant differences between day 1 and 3, “+” between day 3 and day 7 and “#” between day 1 and day 7.