Porphyromonas Gingivalis Biolm Formation on an Implant Surface Treated With Nanoselenium

Background: Biolm formation on implants is the primary factor for implant loss. Porphyromonas gingivalis is a highly virulent pathogen that contributes to the development of periodontal disease and implant failure. Objectives: The goals of this study are to investigate the formation of P. gingivalis biolms on nanoselenium-coated implants in vitro and the potential use of nanoselenium for peri-implantitis treatment. Materials and methods: Porphyromonas gingivalis ATCC 33277 was cultured to obtain an in vitro mature biolm on the surface of the Hexacone implant system. The xture was added into an Eppendorf tube and placed in a sterile air laminar ow cabinet. An automatic machine learning utility was used to calculate the biolm size on the implant surface from SEM images, and the Trainable Weka Segmentation plugin in Fiji software was employed. Results The SeNPs affected the P. gingivalis biolm (the effect size was 80.17%), and the difference was highly signicant (p 0.000). Conclusion: The use of SeNPs as dental implant coatings presented promising anti-P. gingivalis biolm activity. Clinical relevance:: The development of a dental implant surface treatment with ecient antibacterial properties, especially against the most virulent pathogens, has not yet been established. Nanoselenium particles an implant surface coating prevented Porphyromonas gingivalis biolm formation to a striking extent. Nanoparticles could provide a novel state-of-the-art therapeutic approach for Porphyromonas gingivalis (P. gingivalis biolm on dental implants)


Introduction
Bio lm formation on implants is a primary factor in peri-implant disease. Porphyromonas gingivalis is the main causative pathogen in periodontitis. This microorganism is also a risk factor for various systemic diseases, such as certain disorders, diabetes, and pulmonary infection. The features of microbes in a bio lm can deviate signi cantly from that of an equivalent organism under planktonic conditions in terms of the rate of growth and gene transcription (1). Clinically, bio lms form on skin, oral mucosa and teeth sometimes cause chronic infection of dental implants (2).
Management of bio lm-related infections brings great challenges in oral implantology because the structure and composition of the bio lm itself offers protection against antimicrobial agents, and regular mechanical bio lm disruption is required to enable surface disinfection. Medications that may remove such bio lms are needed for clinical use. Dental implants have become the rst choice for patients seeking dental restorations. However, implant mucositis and subsequent peri-implantitis impose a great risk for implant quality and patient comfort. Additionally, bone resorption and even implant loosening account for nearly 30% of total implant failure cases (3). Signi cant data have con rmed that bio lm formation on the implant neck is responsible for implant mucositis (4). P. gingivalis has been con rmed as a critical pathogen in peri-implantitis, a periodontitis represented by in ammation of peri-implant soft and hard tissues. This condition may result in dental implant failure(5), (6), and a considerably high prevalence of peri-implantitis (20% to 56%) has been reported (7). Bio lms are complex communities of microorganisms that are adherent to each other and/or to a surface and are encapsulated within a self-produced matrix (8). These organized communities represent a major implant risk because of their invasion and evasion of host defense mechanisms and their decreased susceptibility to antimicrobials (9). Bio lm-mediated resistance is due to impaired penetration of antimicrobials, upregulation of drug resistance genes, and downregulation of metabolic activity of cells included within the bio lm(10), (11). The pathogenicity of P. gingivalis is expressed by an arsenal of virulence factors connected with tissue colonization and damage and hinders host defense mechanisms (12) , (13) . Nutritional interactions are described to play a role regarding the coexistence of P. gingivalis and T. denticola. P. gingivalis provides isobutyric acid, which promotes the growth of T. denticola, while T. denticola produces succinate, which enhances the growth of P. gingivalis; these interactions might explain the nding that P. gingivalis and T. denticola exhibit enhanced planktonic and bio lm growth once they are cultured together compared to monospecies growth (14) , (15). Antibacterial agents that possess activity against P. gingivalis include quorum sensing inhibitors, antimicrobial peptides, and natural sources such as capsaicin from Capsicum plants (chili peppers) µg/mL (16). Selenium, an important element, is critical for health. In humans, selenium is necessary for the synthesis of 25 selenoproteins. The bene cial effects of selenium on the risk of various cancers (lung, liver, colorectal, prostate, esophageal, gastric cardia, thyroid and bladder) has been con rmed (17) , (18). SeNPs have been studied for certain medical uses and as a possible substance for orthopedic implants (19). The power of selenium compounds as antibio lm and anti-in ammatory agents has been con rmed. Nanostructured materials improve osteoblast functions (such as adhesion (20), . proliferation, synthesis of certain bone proteins, and deposition of calciumcontaining minerals) and encourage adequate osteointegration because of the maximized area and roughness (21), (22). In contrast, tailoring the surface of titanium dental implants with antibacterial agents is a critical aim for implantologists and researchers. Antimicrobial coatings inhibit the infection risks of implants, which are the foremost common explanation for reverse surgery. The antibacterial actions of NPs can be classi ed as (1) damaging the cell membrane, causing cell lysis; (2) disrupting protein synthesis; and (3) preventing DNA replication (23) , (24,25). Many different methods are used to assess bio lms. Biological techniques include the following: semiquantitative staining, measurements of dried biomass, protein or DNA quanti cation, and assessments of residual viable organisms. Each method has advantages and de ciencies, but all of them provide only indirect values of the removal e ciency and are susceptible to operator variability (26,27) (28) . Standard optical microscopy, confocal laser scanning microscopy and epi uorescence microscopy (EM) are reliable tools for bio lm analysis. Additionally, scanning electron microscopy (SEM) is a proper instrument not only for intimately viewing the substratum morphology but also for observing bacterial attachment and bio lm formation on abiotic surfaces. Indeed, SEM has been useful within the event of antibio lm materials for biomedical applications (29) (30) .
Scanning electron microscopy (SEM) has been used extensively for qualitative observation of bio lms because of its high resolution and is typically applied in conjunction with biological assays on bacterial bio lms (31); advanced segmentation techniques such as semisupervised machine learning methods are also typically prescribed (32).

Material And Methods
The minimum inhibitory concentration of SeNPs was tested by the microtiter broth dilution method as described by Khiralla (33). The concentrations of SeNPs were 0, 10, 15, 20, 25 and 30 µg/mL. The MIC90 was set as the lowest concentration of SeNPs that inhibited 90% of the growth compared with the positive control. All tests were administered in triplicate (n = 3), and the results were averaged. (34) Nano preparation An actively growing culture was used to prepare subcultures on potato dextrose agar slants, and after 72 hours of incubation at 28 °C, the slant was used as the starting material for titanium nanoparticle synthesis. Fungus was cultivated in a 250 mL ask containing 100 mL of modi ed malt extract-peptone (MGYP) medium. The pH of the medium was set to six, and a rotary shaker was operated at 150 rpm and 28 °C for 72 hrs. After 72 hours, fungal balls of mycelia were dislocated from the culture broth, and fungal mycelia were rinsed with sterile water. The collected fungal mass (15 g wet weight) was resuspended in 100 mL of sterile Milli-Q-Water in a 250 mL Erlenmeyer ask and then shaken (150 rpm) at 28 °C for 62 hrs. Next, the cell-free ltrate was collected and added to Na2SeO3 salt at a concentration of 10 mM (optimum salt concentration from our preliminary experiment). The whole mixture was placed on a shaker (150 rpm) at 28 °C, and the reaction was allowed to occur over 48 hrs.
Bacterial cell culture and bio lm generation Porphyromonas gingivalis ATCC 33277 was able to develop an in vitro mature bio lm on the surface of the Hexacone implant system. The surface was sandblasted, acid-etched during a heat process, and then osmoactively protected. The features of Hexacone implants include an internal 6-or 12-edge, an indoor marginal taper and a US standard internal thread. The implant is composed of sandblasted Ti6AI4V ELI.n (S); each Hexacone xture was placed in an Eppendorf tube and placed in a sterile air streamline ow cabinet. Each Eppendorf tube was opened and lled with 0.5 mL of a previously prepared P. gingivalis suspension, shaken and then incubated at 37 °C for 3 weeks.

Microscopic examination
Sample Preparation: 1-The samples were xed with 2.5% glutaraldehyde and dehydrated by serial dilution with ethanol with agitation using an automatic tissue processor (Leica EM TP, Leica Microsystems; Austria). 2-Then, the samples were dried using a CO% juncture drier (Model: Audosamdri-8 1 5, Tousimis; Rockville, Maryland, USA). 3-The samples were coated by a gold sputter coater (SPI-Module, U) Microscopic Examination: The samples were investigated by scanning microscopy (JSM-5500 LV; JEOL Ltd. -Japan) under high vacuum at the Regional Center of Mycology and Biotechnology, Cairo, Egypt (Figs. (2, 3, 4)) Three groups were assigned: one group was P. gingivalis bio lm on Hexacone, the second group was P. gingivalis and 25 mg/ml nanoselenium particles, and the third group was a plank Hexacone xture. Each group comprised seven SEM microphotographs. The micrographs were processed using Fiji software.

Visualization of selenium coating and bacterial bio lm
The dental implants were obtained, and the covered area was calculated with the Fiji image processing freeware (National Institutes of Health, Bethesda, Maryland). The bio lm was segmented from the titanium surface in SEM images using an automatic machine learning process (Figs. (5, 6)). The Trainable Weka Segmentation plugin was utilized to classify the titanium surface, the bio lm and the SeNPs and subsequently segment the image; the information was tabulated and statistically evaluated using SPSS 25 software. Ethics approval was not required for this in vitro study.

Results
The rst group (group 1, P. gingivalis only) and the second group (group 2, P. gingivalis + SeNPs) were compared rst. A Shapiro-Wilk test (P = 0.01 and 0.04 < 0.05) for both Group 1 and Group 2 (and a visible inspection of their histograms, normal Q-Q plots, P-P plots and box-plots, Fig. (7)) showed that the data were not normally distributed for either Group 1 or Group 2. Therefore, the nonparametric Mann-Whitney test was used rather than the independent t-test.
Effect sizes are reported using the eta square (η2) index from main and interaction effects. Eta square is de ned as the proportion of variance within the variable that is explained by the study experimental variable.
To test the hypothesis that Group 1 (mean, SD) and Group 2 means (mean, SD) were equal, a nonparametric Mann-Whitney test was performed. Before conducting the analysis, the normality distribution of every group was examined, but was not observed to be satis ed, as mentioned before. The null hypothesis of equal means of every group was rejected, Z = 2.121, P < 0.0001. Thus, the Group 1 mean was statistically signi cantly above the Group 2 mean. The effect size was estimated (35); the results showed a large effect size of η2 = 80.17%, which suggests that 80.17% of the variance of variable SeNPs was predictable from the 2 groups of P. gingivalis when all of the opposite variables were held constant.
Microphotographs of P. gingivalis bio lms without selenium nanoparticles (Fig. 5) and in the presence of selenium nanoparticles are presented (Fig. 6). The SeNPs affected P. gingivalis bio lms (the effect size was 80.17%), and the difference was highly signi cant ( p 0.000), as shown in Figure (8).

Discussion
This study was the rst in vitro study to evaluate the consequences of SeNPs on a major pathogenic cause of periodontitis and peri-implantitis, i.e., P. gingivalis bio lm formation. As a major etiologic cause of the onset of periodontitis and peri-implantitis (36), the bio lm is extremely di cult to eliminate because extracellular polymeric substances with polysaccharides shield pathogens from antibacterial agents (37). This study shows that 25 mg/ml SeNPs reduced P. gingivalis bio lm formation. The inhibitory effect of SeNPs could also be enhanced by increasing the SeNP concentration; the surface should have certain properties to achieve the best rate of osseointegration and combat bacterial challenges, including maintaining host system competence, promoting host tissue integration, inhibiting microbial adhesion and growth and eliminating all organisms on and around the implant (38). It was also found that P. gingivalis proliferation was much lower on nAg-HA/TiO2-coated surfaces than on the uncoated controls (39). It was further found that surface treatment with certain molecules containing antimicrobial and Ti-conjugated peptides may help prevent bio lm formation on Ti surfaces(40). Chimeric peptides represent a favorable alternative to prevent bio lm formation on titanium surfaces, with the promise of stopping peri-implant disease(41).(42).
This consideration is also supported by a study of coCrMo with a 2.5-µm zirconium nitride top coat, which appeared to be a promising surface modi cation technology that has the ability to inhibit bacterial attachment on the surface of an implant and further prevent implant infection with S. epidermidis bio lm formation (43) .

Conclusions
SeNPs provide a helpful method to inhibit bio lm formation and have prospects in clinical use as antibacterial agents in oral implantology. The use of SeNPs as an implant coating presents promising anti-P. gingivalis bio lm activity in vitro and will be further explored for future clinical applications. The chances regarding local infection control may positively in uence the result of implant loss and/or dysfunction. This technology will soon be able compete in augmenting the clinical success of dental implants. The test was performed using an agar-well diffusion assay. The well diameter was 6.0 mm using 100 µl of the tested sample. *RCMB; Regional Center for Mycology and Biotechnology. **N.A.: No Activity.