Biocompatibility and antibacterial properties of zirconium nitride coating on titanium abutments: An in vitro study

Abstract

Improving soft tissue attachment and reducing bacterial colonization on titanium abutments are key factors for the long-term maintenance of healthy soft and hard peri-implant tissues. This in vitro study was conducted to compare the biocompatibility and antibacterial activity of four different surfaces: uncoated Ti6Al4V, anodized, and coated with titanium nitride or zirconium nitride. Surface topography was investigated with a high-resolution system for measuring surface finishes. Human gingival fibroblast (HGF) adhesion and proliferation were examined using MTT assay, Scanning Electron Microscopy (SEM) imaging, immunofluorescence analysis and real-time PCR for selected target genes. The hemolysis and AMES tests were performed to assess the chemical compounds' blood compatibility and mutagenic potential, respectively. Antibacterial activity was tested against five bacterial strains isolated from the oral cavity (Streptococcus salivarius, S. sanguinis, S. mutans, S. sobrinus, S. oralis), and the percentage of dead bacteria was calculated. Roughness measurements confirmed a substantial similarity between the surfaces and their compatibility with clinical applications. MTT assay, SEM analysis and immunofluorescence staining showed adhesion and proliferation of HGFs cultured on all the examined surfaces. PCR confirmed that HGFs produced extracellular matrix components efficiently on all the surfaces. No hemolytic activity was detected, and the AMES test confirmed the surfaces' clinical safety. For all tested bacterial strains, biofilms grown on the zirconium nitride surface showed a higher percentage of dead bacteria than on the other disks. The titanium nitride surface inactivated bacterial biofilms, too, but to a lesser extent.

Fig 1. 3D maps and related 2D profiles.

3D maps and related 2D profiles of: a) machined, b) anodized, c) TiN-coated, and d) ZrN-coated disks.

Fig 2. Proliferation of fibroblasts seeded on Ti disks.

(a) MTT assay of HGFs cultured on the different Ti disks for 3, 7, 14 and 21 days shows that cells are vital and able to proliferate on all the surfaces examined. (b) SEM images (1000x magnification) of HGFs grown onto the four Ti disks reveal that cells reached confluence after 14 days from seeding, forming a continuous monolayer, which persisted up to 21 days.

Fig 3. Real-time PCR analysis.

Real-time PCR analysis of cell adhesion and proliferation markers in HGFs cultured on Ti disks for 21 days. Results for each experiment are obtained from triplicate experiments and values are expressed as the mean ± SD.

Fig 4. IF analysis.

IF staining of HGFs grown on Ti disks for 14 days. Upper panels display staining of vinculin (in green); lower panels show labeling of actin filaments by Phalloidin (in red). For both panels, nuclei are stained blue. Images taken at 40x magnification.

Fig 5. Percentage of dead bacteria.

Percentage of dead bacteria in five strains grown on disks after 120 hours of incubation: a) S. oralis; b) S. salivarius; c) S. sanguinis; d) S. mutans; e) S. sobrinus.