4.3.4. Chlorhexidine Group (CHX)

The bacterial contaminated PICN discs were immersed in 3 mL of 0.12% chlorhexidine (CHX) digluconate solution (Sigma Aldrich) and left for 2 min. The treated specimens were later thoroughly irrigated with PBS solution to remove the excess CHX solution.

### 4.3.5. No Treatment (NT)

The fifth group served as control specimens in which none of the decontamination technique was performed and left untreated.

### *4.4. Microbial Analysis*

Immediately after performing therapies, each PICN discs were immersed in 600 µL PBS solution placed in 2 mL Eppendorf tube. The samples were vortexed for 30 s to detach the bacteria from the specimen surface. 100 µL from each Eppendorf tube were transferred to Mueller Hinton broth (100 µL). Subsequently, 20 µL of PBS from each Eppendorf tube was transferred to a microplate well containing 180 µL of Mueller Hinton Broth. A total of 10-fold serial dilutions were performed in 96-well plates followed by the inoculation of 30 µL of the suspension from each well and plated to Brucella agar plates. Incubation of the plates was performed for 72 h in anaerobic conditions, and CFU were counted later. Distinctive colonies were detected using MALDI Biotyper (Bruker Daltonics, Leipzig, Germany) with visual analysis.

### *4.5. Confocal Laser Microscopy*

PICN discs with the attached biofilms were used for confocal microscopy investigation for Live/Dead Bacterial Viability. The viability of bacteria was checked using a confocal laser scanning microscope (CLSM; Fluoview FV 1000, Olympus, Tokyo, Japan). LIVE/DEAD BacLight stain (Invitrogen, Carlsbad, CA, USA) was used after mixing according to the manufacturer's instructions. The PICN specimens were incubated for 30 min in the dark, excessive stain removed and analyzed with CLSM using light emission between 500 and 550 nm with an excitation wavelength of 488 nm and ×100 objective.

### *4.6. Scanning Electron Microscopy*

A random single sample from each group was selected to study the bacterial adherence and topographical alterations on PICN discs under scanning electron microscope (Tescan VEGA3, Tokyo, Japan). The specimens were fixed using 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (NaCac) with pH 7.4 and stored overnight. Subsequently, the samples were treated with 2% osmium tetraoxide and dehydrated in a series of ethanol concentrations (60–100%). The samples were later processed for critical point drying using CO2. The dried PICN discs were taped in double sided copper tape and sputter coated with 5 nm platinum coating. The PICN discs were imaged at 10 kV using through-lens detector (TLD) for secondary electron imaging. Representative images from each group were selected for depiction.

### *4.7. Statistical Analysis*

Statistical data were analyzed using statistical package of SPSS (v22 IBM Corp., Armonk, NY, USA). Normality testing was performed using Kolmogorov-Smirnov test. Alpha level was set at <0.05. The differences between the groups for each periodontal bacterium assessed and overall count of bacteria were compared using ANOVA test. Multiple comparisons were applied using Tukey-Kramer method. The data for bacteria were log transformed using the following formula [29]:

$$\mathbf{L} = \log\_{10} \left( \mathbf{N} + \mathbf{1} \right) \tag{1}$$

Bacterial reduction and their percentages compared to the NT group was computed using the following formula [29]:

$$(1 - \text{T/C} = 100 \times (1 - \text{T/C})^{\circ}) \text{\textdegree } \tag{2}$$

where T = mean value for each tested group and C = NT group.

### **5. Conclusions**

Photodynamic therapy produced superior periodontal bacterial reduction over the surface of PICN. PDT group showed higher reduction for each bacterial species and total counts of bacteria assessed followed by the DL group. Both PDT and DL treatment strategies are effective without producing surface alterations on PICN.

**Author Contributions:** Conceptualization, E.E. and S.S.; methodology, A.A. and M.S.M.; software, A.A..; validation, E.E., S.S. and M.S.; formal analysis, E.E. and M.S.; investigation, E.E. and A.A.; resources, E.E., M.S., A.A. and M.S.M.; data curation, M.S.M. and M.S.; writing—original draft preparation, E.E., A.A. and M.S.M.; writing—review and editing, E.E., S.S. and M.S.; visualization, E.E. and A.A.; supervision, S.S.; project administration, E.E., S.S. and M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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