Introduction
Microbial dental plaque is defined as a type of highly structurally and functionally organized biofilm consisting of bacterial and non-bacterial microbes aggregations surrounded by an extracellular matrix that includes substance from serum, saliva, and blood (1). Periodontitis/peri-implantitis are pathological conditions associated with biofilm in the tissues surrounding teeth/dental implants. Although certain risk factors such as smoking, inadequate oral hygiene, and poorly controlled diabetes mellitus have been identified in the deterioration of periodontal and peri-implant health, prosthetic factors such as improper prosthetic design, unstable occlusion, the violation of supracrestal adherent tissue, uncleansable bridges, over-contoured crowns, and surface properties of prosthetic materials also play an important role in the development of periodontal and peri-implant diseases (2-7). In addition, the amount and composition of biofilm formation may be influenced by the implant and prosthetic material’s chemical and physical properties such as surface roughness and surface energy (8,9).
Polyetheretherketone (PEEK) has a partially crystalline polymer structure and exhibits high-temperature resistance (over
300 °C), high mechanical strength, and good chemical
resistance (10). In addition, the elastic modulus of PEEK material is close to the elastic modulus of human bone and thus, it has been reported that the longevity of PEEK material in the human body is excellent (11). PEEK has been preferred for use as an alternative to metallic implants in the field of orthopedics and traumatology because of its favorable biomechanic properties and high performance (12). It has also been reported that PEEK is resistant to chemicals in aging environments (13).
Many applicable methods such as direct surface modification, nano-level surface modification, and/or various filler additives can be produced to increase the bioactivity and osteoconductive properties of PEEK (14,15). Recently, modified PEEK materials have been used as alternative materials to titanium, zirconium, and metal alloys in various fields of dentistry, such as infrastructure material in fixed partial dentures, components in removable prostheses, temporary abutments, healing caps, and even implant materials (15,16). A modified PEEK, bio high-performance polymer (BioHPP), has been introduced as a novel material with higher biocompatibility. This modified and ceramic-reinforced PEEK is obtained by adding 20% ceramic fillers (aluminum oxide and zirconium oxide) to the PEEK material. The ceramic microparticles are approximately 0.3-0.5 microns in size, (17,18) and the addition of ceramic microparticles has been shown to improve the polishability and stability of PEEK material. The ceramic-reinforced PEEK material can be used to produce higher-quality prosthetic restorations (13,17). Moreover, the addition of filler has been reported to improve hydrophilic properties compared to pure PEEK material (19).
Prosthetic implant dentistry is an area that is constantly open to new modifications and materials such as ceramics, polymers, and modified PEEK/PEEK. At the same time, it would be beneficial for clinicians to know about improved dental materials that inhibit bacterial adherence and delay biofilm formation. Although there has been a limited number of studies to date, the research has focused on comparing PEEK and titanium surfaces with biofilm formation (20-22). This in vitro study was designed to compare microorganism adherence and biofilm formation of Staphylococcus aureus, Acinetobacter baumannii, Enterococcus faecalis, and Candida albicans on pure PEEK and ceramic-reinforced PEEK.
Methods
The sample size was calculated with a software program (G*Power 3.0.1). The minimum total number of specimens was determined as 72 with 0.403 effect size (f), 0.80 power (1-β err probe), and 0.05 significance level (α err probe). Considering the result of the power analysis, a total of 72 specimens were prepared in the current study, as 9 in each group.
The surfaces of pure PEEK (Juvora; Juvora Ltd. Thornton Cleveleys, Lancashire, England) and ceramic-reinforced PEEK (BioHPP; Bredent GmbH, Senden, Germany) materials were prepared using the same processes. Nine pieces of each pathogen and a total of 72 (36 pure PEEK; 36 ceramic-reinforced PEEK) 8 x 8 x 4 mm rectangular samples were obtained from the manufacturer’s prefabricated blocks with computer-aided design and computer-aided manufacturing and a precision cutting tool (Micracut 151; Metkon Instruments Inc. Bursa, Türkiye) at 400 rpm/min under water cooling. The samples were polished with 180,400,600,800,1200,1800 silicon carbide papers (ScanDia Hans P. Tempelmann) respectively under water spray at 25 N pressure in an automatic polishing device (Tegranim-20, Struers, Ballerup, Denmark) for 15 sec. All the samples were then rinsed in an ultrasonic machine (UT-206; Sharp, Osaka, Japan) with distilled water for 5 min, and the residue was cleaned from them. The samples were sterilized by autoclaving at 134 °C and 3 bar with a 60-minute program.
Surface Roughness
The surface roughness of the samples was determined with a profilometer contact surface measurement device (Sutronic S-series, Taylor/Hobson, Lester, England) using a standard diamond tip (tip angle: 90°, tip radius 2 mm) and a cut-off level of 0.25. A total of 5 measurements (3 vertical and 2 horizontal) were made from all samples. The mean roughness value (Ra) was calculated by averaging the values obtained from each sample.
Microbial Cultures
All the test microorganisms used in this study were the American Type Culture Collection (ATCC) strains including; S. aureus (ATCC 29213), A. baummanii (ATCC 19606), E. faecalis (ATCC 29212), and C. albicans (ATCC 10231).
The microorganisms were first inoculated into 5 mL nutrient broth and were incubated at 37 °C for 24 h. Microorganisms from these cultures were transferred onto a solid medium and incubated overnight. Tryptone Soy Agar (Oxoid, UK) was used for S. aureus, E. faecalis, and A. baummanii, and Sabouraud Dextrose Agar (Biolife, Italia) was used for C. albicans. After growth, selected colonies were transferred into a liquid medium and incubated for 4-6 h to achieve log phase growth. Tryptone Soy Broth (TSB) (Oxoid, UK) for bacterial strains and Sabouraud Dextrose Broth (BD, Difco, USA) for C. albicans, were the selected media for this purpose. The optical density of each culture at 490 nm (OD 490) was adjusted to 1-3x108 colony forming units (CFU)/mL.
Biofilm Formation Assay
Biofilm formation was measured with a modification of the method used by Peng et al. (23). One mL of a microbial suspension of S. aureus, E. faecalis, A. baummanii, and C. albicans (106 CFU/mL) was inoculated in all the wells of a 24-well plate. The testing samples (pure PEEK and ceramic-reinforced PEEK) were added to the wells and incubated for 72 hours at 37 °C. At the end of the incubation, the broth medium was removed and the wells were washed twice with phosphate-buffered saline (PBS) and air-dried for 1 h. After adding crystal violet (0.1% w/v) to each well, the plate was allowed to remain at room temperature for 15 min. After 15 minutes, the stain was aspirated and the plate was rinsed four times with PBS. In the last step, 1 mL of 33% acetic acid was added to the wells. Absorbance was determined at 490 nm wavelength (OD 490) on a microplate reader (Biotek, ELx800 Absorbance Microplate Reader, USA). TSB and SDB without microbial suspension were used as negative controls.
Statistical Analysis
Data were analyzed using IBM SPSS V21 software (SPSS IBM, Chicago, IL, USA). According to the Kolmogorov-Smirnov test, the data showed normal distribution (p>0.05). The comparison of the two groups was analyzed with the Independent Samples t-test. Intra-group analyses were performed with a two‐way analysis of variance (ANOVA) followed by Post hoc Bonferroni adjustment. The level of statistical significance was determined as 0.05. The correlation between surface roughness and biofilm formations were analyzed with pearson correlations.
Results
The surface roughness was mean 0.56±0.88 Ra for pure PEEK, and 0.52±0.83 Ra for ceramic-reinforced PEEK, with no significant difference determined between the groups (p=0.054) (Table 1). There were no significant correlations between surface roughness of peek materials and biofilm formation (Juvora; r=-0.190, p=0.266, BioHPP r=-0.018, p=0.916 ) (Table 2, Figure 1). The results obtained from the microbial analysis of S. aureus, E. faecalis, A. baummanii, and C. albicans are shown in Table 2. No significant difference was found in terms of the biofilm formation of the PEEK groups for each pathogen (p>0.05). In each group evaluation, the highest biofilm formation was found in S. aureus, followed by A. baummanii, E. faecalis, and C. albicans in the pure PEEK group (p<0.001). The highest biofilm accumulation in ceramic-reinforced PEEK was recorded in S. aureus and A. baummanii. The biofilm formations of E. faecalis and C. albicans were significantly lower than those of S. aureus and A. baummanii (p<0.001) (Table 3).
Discussion
Microbial dental plaque is considered a primary etiological factor for the development of periodontal disease and should be controlled as the first step in periodontal treatment. The attachment of microorganisms to surfaces occurs through complex chemical and physical mechanisms. The surface and chemical properties such as roughness, hydrophilicity, nano topological structure, and modifications with antibacterial coatings of a prosthodontic or implant material can be effective in inhibiting biofilm covering (24,25). Therefore, it is important to know the properties of prosthodontic biomaterials that enhance biofilm formation. A search of the literature revealed a few studies that analyzed biofilm formation on PEEK surfaces. This investigation aimed to evaluate the biofilm formation of different strains on pure PEEK and ceramic-reinforced BioHHP materials. The second aim of this study was to contribute to the literature by defining the properties of BioHPP. The hypothesis of the study was that there was a difference in biofilm formation between PEEK and ceramic-reinforced PEEK materials.
Previous studies have noted the importance of good mechanical properties of BioHPP such as good marginal quality, fracture resistance, retention, high polishing, low absorption properties, wear resistance, and aesthetics (26-29). Jin et al. (26) suggested that BioHPP, compared to titanium, could be used as an alternative material to be veneered with composite resin due to higher shear bond strength compared to composite resin. Porojan et al. (17) found that BioHPP was less affected by aging than pure PEEK due to the nanosurface topography and nanoroughness. BioHPP is reinforced with ceramic molecules; aluminum oxide, and zirconium oxide (18). Zirconia is known to attract low plaque accumulation (30,31). Therefore, assessing biofilm formation on ceramic-reinforced and pure PEEK materials would be of interest. In the current study, the differences in biofilm formation between pure PEEK and BioHPP were not significant. Wiessner et al. (32) conducted an in vivo study to examine the formation of biofilm on various materials, including titanium, zirconia, PEEK, and PEEK-BioHPP. They employed fluorescence microscopy and image analysis software to quantify the biofilm formation. Their findings indicated that zirconia exhibited the least biofilm formation, followed by titanium, PEEK, and PEEK-BioHPP. Their observations align with the outcomes of our study, as no difference in biofilm formation was observed between PEEK and modified PEEK materials.
Higher surface roughness can influence bacterial attachment, increasing the surface area, and causing unsmooth and uncleanable surfaces (9). A key strength of the present study was that no difference was determined in the surface roughness between the PEEK groups. In a recent study, Barkarmo et al. (22) found that while there were no significant differences in biofilm adhesion of streptococci strains between smooth PEEK and titanium surfaces, blasted PEEK material had significantly more biofilm formation. Hahnel et al. (33) reported in a laboratory study that biofilm formation of Streptococcus gordonii, Streptococcus mutans, Actinomyces naeslundii, and C. albicans on the surface of PEEK from zirconia and titanium abutment materials was equal or less. In addition, the surface roughness of PEEK surfaces was also found to be lower than that of zirconium and titanium abutment surfaces. In addition, one study found biofilm formation on ceramic-reinforced PEEK, BioHPP. Similarly, in this study, the adhesion of S. aureus and C. albicans on dental polymers was mainly affected by surface roughness (34). These results might be explained by the fact that the surface roughnesses were different between blast, rough, polished, and smooth surfaces and increased surface roughness had an impact on the biofilm adhesion.
In the current study, tests were made of four different biofilm-producing strains of microorganisms, namely S. aureus, E. faecalis, A. baummanii, and C. albicans, on pure PEEK and ceramic-reinforced PEEK material obtained by adding 20% ceramic (aluminum oxide and zirconium oxide). S. aureus is an important pathogen of implant-associated osteomyelitis infections (35,36). Candida spp. have been associated with denture stomatitis (37). E. faecalis acts as an important agent in oral infections and A. baummanii is a multidrug-resistant bacteria of clinical importance (38,39). The present study showed considerably high S. aureus accumulation and biofilm formation on both biomaterials when compared with the other pathogens. In contrast to S. aureus, E. faecalis and C. albicans strains formed fewer accumulations. Although not statistically significant, A. baummanii formed more biofilm on the ceramic-reinforced BioHHP group than on pure PEEK surfaces. These results led to a review of the previous studies that investigated the antimicrobial properties and biofilm-forming characteristics of aluminum oxide and zirconium oxide, which were added to PEEK material.
Compared to the nanoparticles of oxides of metals such as silver, iron, zinc, and copper, there have been few investigations of the antimicrobial efficacy of aluminum oxide nanoparticles. According to the results of a recent review, studies about the inhibitory effect of aluminum oxide nanoparticles have reported that aluminum oxide nanoparticles cause a decrease in the growth rate of S. aureus and C. albicans, as well as show an inhibitory effect at moderate concentrations and a bactericidal effect at high concentrations for A. baumanii. Compared to other metal oxide nanoparticles, aluminum oxide nanoparticles have the lowest microbial inhibitory effect (40).
Zirconia has also been used introduced to the field of dentistry due to mechanical stability and it can be used as pure zirconia or alumina-toughened zirconia (41). To date, limited scientific data regarding bacterial accumulation around zirconia dental implants and abutments are available and the results are conflicting. Studies investigating zirconia implants have shown less oral biofilm in vivo and conversely, more biofilm in vitro when compared with titanium implants. As these data can not allow a clear preference for the use of zirconia, more studies have been carried out to provide further information (42). Zeller et al. (43) investigated biofilm formation on discs of metal alloys, zirconia, and PEEK in vivo and detected higher biofilm mass formation, and the zirconia and PEEK levels were similar. Roehling et al. (44) compared biofilm formation on zirconia and titanium implant surfaces and detected a statistically significant reduction in biofilm formation. Abualsaud et al. (45) evaluated the antimicrobial effects of zirconium dioxide nanoparticles reinforcement of poly(methyl) methacrylate on surface roughness and C. albicans biofilm and an insignificant reduction of C. albicans biofilm formation was observed.
Study Limitations
The results of the current study, indicating no difference between the two tested biomaterials by means of microbial attachment, can be interpreted through comparisons with the reports of some former studies concerning low antimicrobial efficiencies and the biofilm formation inhibitory effects of zirconia and aluminum oxide. However, as zirconia and aluminum oxides are mixed with PEEK to obtain ceramic-reinforced BioHHP material, interpretation is not straightforward. These conflicting results can be associated with the nature of the machined or modified PEEK surfaces. Moreover, biofilm involvement can be affected by many clinical factors such as the plaque removal efficiency of the patient the location of restorations, and improper finishing/polishing of restorations. Therefore, conducting our study under in vitro conditions is one of the limitations of our study. The certain limitation of this study was the surfaces of PEEK were standardized with silicon carbide papers and the effect of different surface modifications on biofilm formation on the surface of the PEEK materials was not evaluated. The effect of different surface modifications on biofilm formation on PEEK surfaces should be evaluated in further investigations. Another limitation was that the study evaluated only two specific PEEK materials, whereas there were various PEEK materials in the dentistry field. This study was a preliminary study to define the biological characteristics of ceramic-reinforced PEEK. Biofilm retention of PEEK materials could be evaluated with further clinical studies, and comparative evaluation of the effects of various PEEK materials on biofilm formation would be effective in obtaining comprehensive findings.
Conclusion
The study results showed no difference in terms of biofilm formation between pure and ceramic-reinforced PEEK materials. It can be suggested that the association of biofilm formation on modified PEEK materials be investigated in future clinical studies.
Ethics
Ethics Committee Approval: Ethics committee approval is not required.
Informed Consent: Informed consent is not required.
Authorship Contributions
Surgical and Medical Practices: S.K.Y., K.A., B.K., S.S., A.D.K., Concept: S.K.Y., K.A., B.K., S.S., A.D.K., Design: S.K.Y., K.A., S.S., Data Collection or Processing: S.K.Y., K.A., S.S., Analysis or Interpretation: S.K.Y., K.A., K.D., Literature Search: S.K.Y., K.A., A.D.K., K.D., Writing: S.K.Y., K.A., A.D.K., K.D.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.