Fig 1.
A peristaltic pump is used to obtain a continuous flow of nutrients over the surface of the tested specimens. Culture broth is then discarded.
Fig 2.
A: Individual mandibular thermoformed acrylic customized tray obtained using a study cast of the lower jaw. B: On the buccal part of the device three half-implants, one for each experimental surface, were fixed horizontally on each side (three on the right and three on the left) using orthodontic 0.1 mm wire.
Fig 3.
SEM analysis of the in vitro tested surfaces prior to microbiological procedures.
A, B, C, and D, show 500X magnifications of machined, grit-blasted, laser-treated and enamel surfaces. E, F, G, and H, show the same surfaces at 5000X magnification. Grooves were present on the machined titanium surfaces, grit-blasted specimens showed characteristic surfaces with high SR, and laser-treatment left very regular microtextured surface with pits of 18–20 μm diameter.
Table 1.
Surface roughness (Ra) and total surface free energy of the tested titanium surfaces and reference surface (human enamel).
Fig 4.
Sessile drop method for SFE evaluation.
A 6 μl drop of HPLC-grade ultrapure water was photographed (EOS 500D camera, EF 100mm 2.8f macro lens and Speedlite 470EX bounced flash, Canon, Tokio, Japan) on the surfaces of the experimental specimens as follows: A, machined; B, grit-blasted; C, laser-treated; D, enamel. The hydrophobic behavior of grit-blasted and laser microtextured titanium surfaces can be clearly seen compared to machined ones. The hydrophobic behavior of laser-treated surfaces is likely due to a lotus leaf effect. Hydrophilic enamel reference surfaces are also shown.
Fig 5.
Biofilm formation, assessed by MTT assay.
Data are shown as means +/- 1 SD. In each study, different superscript letters highlight significant differences between groups (p<0.05). A: in vitro data showed that laser-modified surfaces were less colonized than grit-blasted ones. Machined surfaces showed intermediate values of biofilm formation, non-significantly different from grit-blasted or laser-treated surfaces. Enamel reference surfaces showed similar biofilm formation as grit-blasted surfaces. B: machined and laser-treated surfaces were less colonized than grit-blasted ones, while no significant differences were identified between machined and laser-treated surfaces.
Fig 6.
SEM analysis of the tested surfaces after in vitro biofilm formation.
A, B, C, and D, show 500X magnifications of machined, grit-blasted, laser-treated and enamel surfaces. E, F, G, and H, show the same surfaces at 5000X magnification. Grit-blasted and enamel specimens showed complete surface coverage by a multi-layered biofilm. Machined surfaces showed large microcolonies covering a minor part of the surface, and laser-treated surfaces showed the least biofilm formation.
Fig 7.
SEM and acquired elemental maps from EDS analysis.
A, B: machined surfaces (titanium displayed in red) showed sparse, dark-gray islets made of carbon (in green) that can be regarded as residues of the machining procedures, incompletely removed by subsequent cleansing. C, D: grit-blasted specimens examined after biofilm formation (highlighted by carbon presence, in red) showed aluminum traces (blue, arrows) corresponding to alumina particles embedded into the titanium surface (green) and still present after biofilm formation. E, F: Enamel surfaces (calcium in green) covered by biofilm structures (carbon in red). No contaminating agents derived from polishing procedures can be observed. G, H: high magnification of grit-blasted and laser-treated specimens, respectively, after biofilm formation. In laser-treated specimens, microorganisms show a colonization pattern where cocci, bacilli and extracellular matrix residues can be found on the edges of the pits. Reduced or no biofilm formation can be seen in the inner part of the pits, which were mostly colonized by streptococcal forms.
Fig 8.
CLSM reconstructions of in vitro biofilm formation.
A, B, C, and D, show 3D reconstructions of machined, grit-blasted, laser-treated and enamel surfaces. E shows a MIP of laser-treated surface, where the titanium surface was acquired on a separate channel and displayed in gray. In all reconstructions, viable microorganisms are stained in green, while dead microorganisms are stained in red. Compact biofilm structures with a prevalence of viable cells covering most of the machined, grit-blasted and enamel specimens can be observed. Laser-treated surfaces displayed less dense biofilm structures mainly constituted by microcolonies of approximately 200 μm diameter.
Table 2.
Calculations of total surface coverage, and percentage of viable and dead bacteria in the biomass, resulting from CLSM MIP images.
Fig 9.
SEM analysis of the tested surfaces after in situ biofilm formation.
A, B, and C, show 500X magnifications of machined, grit-blasted, and laser-treated surfaces, respectively. E, F, and G, show the same surfaces at 5000X magnification. A similar biofilm formation pattern as in the in vitro experiment can be observed.
Fig 10.
CLSM reconstructions of in situ biofilm formation.
A, B, and C, show 3D reconstructions of machined, grit-blasted, and laser-treated surfaces, respectively. A complete coverage of the inner parts of the implant threads with mostly viable cells can be observed in all specimens except for laser-treated surfaces. D (grit-blasted specimen) and E (laser-treated specimen) reconstructions show at a higher magnification the inner part of an implant thread. Laser-treated specimens present few dead microbial cells colonizing the bottom of the threads, while an intense colonization of predominantly viable cells can be found on the inclined surfaces of the threads.