Microbiological and Imaging-Based Evaluations of Photodynamic Therapy Combined with Er:YAG Laser Therapy in the In Vitro Decontamination of Titanium and Zirconia Surfaces

Abstract

The oral cavity's soft and hard tissues create a conducive environment for microbial proliferation and biofilm development, facilitating the colonization of prosthodontic and implant materials such as titanium (Ti) and zirconia (Zr). This study aimed to compare the efficacy of conventional decontamination methodologies (i.e., chemical and mechanical, using 0.12% digluconate chlorhexidine (CHX) solution-treatment and airflow) to adjunctive laser-based interventions on Ti and Zr substrates inoculated with Staphylococcus (S.) aureus ATCC 25923. Additionally, this investigation sought to elucidate the impact of these treatments on temperature variations and surface integrity, analyzing the laser irradiation effects on these prevalent dental materials. Experimental configurations were delineated for both Ti and Zr samples across four groups: (1) a conventional treatment group (CV); (2) a photodynamic therapy group (PDT); (3) an Er:YAG laser treatment group (Er); (4) a combined PDT and Er:YAG treatment group (PDTEr). Also, a negative control group (C) that received no treatment was considered. The decontamination of the inoculated disc samples was evaluated by quantifying the microbial colonies in colony-forming units per milliliter (CFU/mL). Temperature variations on the surface of the samples were determined during laser treatments. Surface modifications were investigated using scanning electron microscopy (SEM) and optical coherence tomography (OCT). For statistical analysis, Fisher 95% confidence intervals, Hsu's MCB method, and the Kruskal-Wallis test were applied. With regard to the 10⁵ CFU/mL of the negative control group, results indicated average values equal for each study group to (1) 2.66 CFU/mL for Ti and 2 CFU/mL for Zr for the CV group; (2) 0.33 CFU/mL for Ti and 1 CFU/mL for Zr for the PDT group; (3) 1.25 CFU/mL for Ti and 0 CFU/mL for Zr for the Er group; (4), and 0 CFU/mL for both Ti and Zr for the PDTEr group. Therefore, the combined PDT and Er:YAG treatment (PDTEr) and the singular PDT modality outperformed conventional decontamination methods in eradicating S. aureus biofilms from both Ti and Zr surfaces. Notably, the PDTEr regime achieved a comprehensive elimination of microbial colonies on treated substrates. Surface examination employing OCT demonstrated discernible alterations in the surface morphology of samples subjected to Er:YAG and combined PDT and Er:YAG treatments. Temperature checks during treatments showed no major changes, suggesting the applied laser methods are safe. In conclusion, PDTEr and PDT eliminated bacteria more effectively, but Zr surfaces were more resilient, making them better for microbe-controlling applications. Also, the study demonstrated that the (less costly but lower resolution) OCT method can replace SEM for such investigations.

Keywords: Er:YAG laser; Staphylococcus aureus; biofilm; dental implants; laser therapy; optical coherence tomography; photodynamic therapy; scanning electron microscopy; titanium; zirconia.

Figure 1

(a) Sterile Ti and Zr discs; (b) densitometer utilized for the suspension of S. aureus ATCC 25,923 0.5 Mc Farland; (c) harvesting S. aureus from Columbia Agar medium and checking the concentration of 0.5 CFU/mL using the McFahrad densitometer; (d) tubes with 4 mL of S. aureus suspension, having the samples immersed in a saline solution.

Figure 2

Surface irrigation with 2 mL of CHX 0.12% solution, followed by decontamination with airflow powder for 10 s using a PROPHY Mate M4 Prophylaxis Handpiece and KAVO PROPHYflex Prophylaxis Powder Perio Powder.

Figure 3

The 635 nm laser diode irradiation in 3 cycles of 10 s each by brushing movements at the level of the treated surface, with 10 s break between 2 successive irradiation cycles.

Figure 4

Irradiation with the Er:YAG laser through brushing movements at the level of the treated surface, oriented at an angle of 45°, and positioned at approximately 3 mm from the surface.

Figure 5

(a) Monitoring temperature variations during the laser treatment using a thermal camera FLIR T640 at (b) the level of the sample treated using both PDT and the Er:YAG 2094 nm laser.

Figure 6

(a) Harvesting with a sterile swab from the treated surface of the samples; (b) storing of the culture media.

Figure 7

(a) Inoculation of the culture media for each individual sample; (b) the presence of two microbial colonies developed on the culture medium after 24 h of incubation at the thermostat for a sample collected from a Ti disc; (c,d) the number of colonies developed on the culture medium for the negative control samples quantified as 10⁵ CFU/mL.

Figure 8

SEM images of Ti samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for a 30× magnification in column (1), as well as for a 130× magnification in column (2).

Figure 8

SEM images of Ti samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for a 30× magnification in column (1), as well as for a 130× magnification in column (2).

Figure 9

SEM images from Zr samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for a 30× magnification in column (1), as well as for a 130× magnification in column (2).

Figure 9

SEM images from Zr samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for a 30× magnification in column (1), as well as for a 130× magnification in column (2).

Figure 10

OCT images for Ti samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for the 5 × 5 mm investigated area in column (1), as well as for the 3 × 3 mm investigated area in column (2).

Figure 10

OCT images for Ti samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for the 5 × 5 mm investigated area in column (1), as well as for the 3 × 3 mm investigated area in column (2).

Figure 11

OCT images for Zr samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for the 5 × 5 mm investigated area in column (1), as well as for the 3 × 3 mm investigated area in column (2).

Figure 11

OCT images for Zr samples presented as examples from each study group: (a) CV, (b) Er:YAG QSP mode, (c) Er:YAG SSP mode, and (d) PDT and Er:YAG for the 5 × 5 mm investigated area in column (1), as well as for the 3 × 3 mm investigated area in column (2).

Figure 12

SEM images, where diameters and radiuses of two samples were measured for (a) Ti and (b) Zr.

Figure 13

Example of SEM images acquired for measurements: (a) entire sample; (b1–b4) quadrants of the same sample.

Figure 14

Results of the assessment of microbial colonies for each of the study groups (i.e., CV, PDT, Er, and PDTEr), quantified in colony-forming units per milliliter (CFU/mL): (a) total CFU/mL in each treatment group; (b) average CFU/mL for each treatment group, the latter calculated as the ratio between the total CFU/mL for each group (indicated on top of the bars in (a)) and the number of considered samples.

Figure 15

Example of SEM images with measurements: (a1–a4) quadrants of a sample.

Figure 16

Example of OCT images with measurements: (a1,a2) halves of a sample.