Filling Materials Efficacy on Preventing Biofilm Formation Inside Screw Access Channels of Implant Abutments

Many different materials can be used to fill screw access channels in implant-supported prostheses. The most frequent materials used are cotton pellets under provisional methacrylate and gutta-percha under composite resins.¹  Recently, polytetrafluoroethylene (PTFE) tapes were also cited to be used under composite resins for final restorations.²  The indications for using these materials are based on that (i) they can be sterilized, (ii) the ease of manipulation or removal when required,¹  and (iii) esthetics.^3,4 

However, it is important to consider that after being placed in the oral cavity, these materials are susceptible to biofilm formation, due to bacterial colonization on the internal surfaces of implant systems,^5,6  thus influencing the prevalence of microbial species in the peri-implant sulcus.⁷  Considering this, screw access channels in implant-supported prostheses represent a microbiologic complication to the dentist. In vitro^8,9  and in vivo^7,10  studies have shown bidirectional fluid and bacterial leakage into and out of implant-abutment junctions.^11,12 

Candida spp., especially Candida albicans, is one of the most common microorganisms present in the oral cavity of denture wearers, affecting 24% to 60% of individuals.¹³  This highlights the importance of C. albicans for in vitro biofilm research related to implant-retained prostheses with large acrylic resin surface areas.¹⁴  The oral cavity is an optimal environment for microbial colonization and consequent biofilm formation due to a vast number of retentive areas¹⁵  and supply of nutrients.¹⁶ 

Recent studies have evaluated levels of microleakage through the screw access channels in implant-supported prostheses sealed with different materials. Park et al¹⁷  used a basic fuchsine dye to demonstrate that there was lower microleakage associated with the use of gutta-percha than with cotton pellets. Nascimento et al¹⁸  showed that none of the tested sealing materials prevented microbial leakage, despite the lowest values for PTFE tape associated with composite resin or gutta-percha and highest microbial counts for cotton pellet associated with light-polymerized provisional composite. Another in vitro study, recently published by Cavalcanti et al,¹⁹  showed a lower leakage in specimens filled with gutta-percha compared with others filled with PTFE. However, these results are contrary to those of Alshehri and Albaqiah,²⁰  who suggested that gutta-percha should not be considered a material of choice.

Currently, the choice of which material to use to fill screw access channels in implant-supported prostheses basically depends on the operator's preference and is influenced by clinical requirements. This method of determining which material to use is seldomly supported by science-based dental implant studies.^1,20–22  This emphasizes the need for a microbiological investigation of the various materials used to fill screw access channels in implant-supported prostheses and their possible influence on biofilm formation.

The occurrence of bacterial colonization on the internal surfaces of implant systems,^5,8,9,23  with differing implant-abutment connections^26,27  and applied tightening torques²⁸  has been shown by in vivo^7,10  and in vitro studies,^24,25  The aim of this in vitro study was to determine and compare the total amount of C. albicans biofilm on various materials used to fill screw access channels in prosthetic implant abutments. The null hypothesis states that there would be no difference in the total amount of biofilm formation between tested materials.

This experimental study consisted of an in vitro, blind, and randomized analysis of different materials used to fill screw access channels in implant-supported prostheses. Forty-five titanium external hexagon connection analogs Ø4.1-mm diameter had their respective titanium abutments (Neodent, Paraná, Brazil) screwed at 32 Ncm, according to the manufacturer' specifications. Then they were divided into 5 groups, according to the materials tested: cotton, gutta-percha,²⁹  or PTFE tape² ; positive control group (with no filling material); and negative control group (closed with light polymerized temporary resin, Clip F; Voco GmbH). The characteristics, manufacturers, and batch numbers of materials used are shown in Table 1.

All tested materials were sterilized for 30 minutes at 121°C with 9.8 N/cm² pressure. Then, in a laminar air-flow chamber (aseptic environment), the abutments of test groups were filled with evaluated materials, vigorously condensed to 3 mm below the border of the titanium abutments (space measured with a cursor fixed to the condenser and endodontic ruler). Gutta-percha and PTFE were cold condensed, and cotton was weighed to be assured that the same amount of material would be used in each specimen, due to the difficulty of standardizing the strength of condensation. In the negative control group, the temporary resin was light-cured for 40 seconds using a Demetron Optilux 401 device (Kerr Corporation, Orange, CA).

Next, they were immersed in a brain heart infusion (BHI) medium containing C. albicans (strain 10231 from American Type Culture Collection [ATCC]) and stored aerobically at 37°C with gentle agitation. Culture media were changed every 48 hours. After 15 days, the materials were removed from the abutments, and the biofilm on the materials and screws was quantified by methyl tetrazolium (MTT) reduction assay at 490 nm. All experiments, considering the 5 groups, were performed in triplicate.

C. albicans (ATCC 10231) was reactivated in BHI (Difco, Tucker, GA) medium for 48 hours at 37°C with gentle agitation. Next, cells were harvested by centrifugation, and the pellet was washed twice with phosphate-buffered saline (PBS). Finally, cells were counted in a Neubauer chamber (Boeco, Hamburg, Germany), allowing the determination of a standardized C. albicans suspension with 3 × 10⁶ cells/mL in BHI medium.³⁰ 

Titanium analogs and abutments with tested materials of all 5 groups were then completely immersed in 5 mL of the suspension in 6-well cell culture plates (Zellkultur Testplatte 24) and incubated aerobically at 37°C with gentle agitation for 14 days.^12,28  Every 2 days, the culture medium was removed, and the nonadhered and dead cells were washed 2 times with PBS. Then, new culture medium was added to restore the initial volume of 2 mL, renewing the nutrients and reestablishing the pH.

Quantification of C. albicans biofilms was performed by analysis of cell metabolism, using the methyl tetrazolium [MTT; Sigma Chemicals, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] reduction assay.^31,32  Briefly, MTT was prepared as a saturated solution at 5 mg/mL, filter sterilized through a 0.22-mm pore-size filter, aliquoted, and stored. An aliquot of the stock solution of MTT was dissolved in PBS at a final concentration of 1 mg/mL. For each assay, the MTT solution was mixed with menadione solution (0.4 mL; Sigma-Aldrich, St Louis, MO) at a volume ratio of 20:1.³⁰ 

After 14 days of incubation,^12,28  titanium analogs and abutments were removed from the C. albicans suspension and washed twice with 1.0 mL sterile PBS solution to remove nonadherent cells.³³  Afterward, the filling material was removed, submerged in 500 μL of MTT–menadione solution, and vortexed to allow complete biofilm contact with the solution, followed by 4 hours incubation in the dark at 37°C. In the end, the formazan precipitate was solubilized in 500 μL dimethyl sulfoxide (DMSO; Sigma Aldrich) and incubated for another 1 hour in the dark at room temperature.^29,30 

The MTT–menadione solution was also used on the screw (after material removal) to evaluate whether there was biofilm formation close to the abutment-analog junction, an indication of C. albicans growing through the entire material; for this purpose, the positive and negative control groups were used as parameters.

Cell viability quantification was evaluated by spectrophotometry at a wavelength 492 nm^29,30 with an Enzyme Linked ImmunonoSorbent Assay (ELISA) microplate reader (model 3550-UV; Bio-Rad Laboratories, Sigma). Values obtained from the experiments performed in triplicate were averaged and expressed in optical density (OD).

The sample size was calculated with G.Power 3.1.9.4 (Kiev University, Germany), using an effect size of 0.7, previously established in a pilot study, with an α error at 0.05 and power at 0.80. IBM SPSS Statistics v.25 was used to calculate normality and comparisons between groups. Normality of the distribution was tested with the Kolmogorov-Smirnov test for each group (G1 = 0.200, G2 = 0.08, and G3 = 0.200). One-way analysis of variance (ANOVA) and Bonferroni post hoc tests were used to analyze differences between groups, with an overall significance level of .05. The methodology and results were reviewed by an independent statistician.

The quantification of C. albicans biofilm formed on the tested materials, expressed in OD, is shown in Figure 1. Analysis of the MTT assays reflected the presence of cell viability, so that the higher the OD value, the larger the quantity of biofilm formed.

The 1-way ANOVA test showed that differences in the mean values among the tested groups were higher than would be expected by chance; there was a statistically significant difference for cotton group (P < .001). The Bonferroni post hoc test demonstrated a significant difference between the means of the cotton and gutta-percha groups (P < .017) and the cotton and PTFE groups (P < .025). However, there was no difference between the gutta-percha and PTFE tape groups (P > .050). Table 2 shows the statistical comparison and statistical significance for each group.

Considering the OD values for biofilm formation on the screw, an indication of C. albicans growing through the material, only the group filled with cotton presented biofilm formation on the screw surface, similar to the control group (data not shown), which did not receive any filling material. The negative control closed with light polymerized temporary resin did not allow any biofilm formation on the screw, similar to the gutta-percha and PTFE tape groups.

The choice of material to fill screw access channels in implant-supported prostheses and to protect the screw is an important issue, not only for esthetics and easy removal reasons,^1,2  but also for biofilm formation, because these materials can act as possible bacterial reservoirs. This entailed the microbiological investigation conducted in this study, with the aim of determining and comparing the total amount of C. albicans biofilm formation on cotton pellet, gutta-percha, and PTFE tape, by gaining entry through the abutment access. These materials were tested because they are commonly used to fill screw access channels in implant-supported prostheses.

Although there are other filling materials, such as vinyl polysiloxane and polymerizing acrylic resin, they cannot be sterilized¹ ; therefore, they were not tested in the present study. Also, with the use of a polymerizing acrylic resin, there is the risk of damaging the screw head during resin removal.¹  Thus, clinicians must consider all material characteristics before choosing the one to use for filling screw access channels in implant-supported prostheses.

The screw access channels of all titanium abutments (except the negative control group) were left open to standardize the biofilm access to the tested materials condensed inside the abutments. In this situation, only the properties of each material, after they were condensed, were tested considering their ability to permit biofilm infiltration and proliferation through the abutment up to the screw connection base. The decision to do this for this present study was similar to the study of Raab et al,²²  where sealing with composite was not done to simulate consistent conditions and ensure the homogeneous growth of bacteria and Candida.

C. albicans was used because it is the most common microorganisms present in the oral cavity of denture wearers.¹³  Therefore, C. albicans has importance for in vitro biofilm research related to implant-retained prostheses with large acylic resin surfaces.¹⁴  The acrylic resin is an optimal environment for microbial colonization due to a vast number of retentive areas¹⁵  and supply of nutrients.¹⁶ 

The null hypothesis was rejected. There were differences in microbiological behavior among the tested materials used to fill screw access channels in implant-supported prostheses after they were vigorously condensed inside the titanium abutments and exposed to a microorganism suspension.

Some previous studies corroborate with this present study.^17,18,22  Nascimento et al¹⁸  microbiologically evaluated PTFE and cotton, as spacer materials in extracted human molars, to replicate the situation after endodontic treatment. The results obtained were that cotton spacers (unlike PTFE tape) may permit bacterial leakage into access cavities and serve as a route for bacterial contamination into the root canal space. Park et al¹⁷  evaluated microleakage using basic fuchsine dye through the screw access channels in implant-supported prostheses sealed with different materials, which also revealed that there was lower microleakage associated with gutta-percha than with a cotton pellet. The study of Raab et al²²  evaluated colonization of Streptococcus mutans, oralis, and C. albicans on sealing materials. They found that cotton had a higher potential of bacterial and fungal adhesion and thus was ineffective for sealing the screw channel, whereas PTFE and gutta-percha could be suggested as materials of choice.

These results may be due to the plastic nature of gutta-percha,²⁷  which allows its insertion, condensation, and filling of empty spaces, thus promoting more efficient sealing, in a similar way as PTFE tape. However, our results show higher values for standard deviation for PTFE tape, which probably correlates with inconsistent condensation. This phenomenon highlights the importance of good condensation if PTFE is selected. Regarding the results for cotton, this material presents a fibrous structure that provides an ideal niche for bacterial growth and material degradation. In addition, cotton cannot be compacted with the same density as gutta-percha or PTFE. The cotton allows for dead spaces between filaments where bacteria may thrive.^22,34  This is an important issue because the cotton pellet is one of the most frequently used materials for filling screw access channels in implant-supported prostheses.^1,3,4 

In view of this, materials used to fill the screw access hole of implant-retained prostheses are susceptible to contamination when exposed in the oral cavity. It is necessary to establish microbiological parameters to aid in material choice. Depending on the microbiological behavior, the material may not only provide sealing capacity but also prevent the accumulation of microorganisms within the implant system.^1,2  Thus, the results of the present study can provide support for future studies. Future studies should evaluate situations that can increase the occurrence of gaps and bacterial growth inside the implant/abutment connection and screw channels,¹²  such as dynamic loading conditions^17,23  and repeated loosening and re-tightening of abutment retaining screws.^5,12,26  Beside the need for long-term clinical analyses, clinical studies must confirm the clinical viability of the findings from this present study. Clinical studies should evaluate both contamination and the physical properties of these filling materials used in screw access channels in implant-supported prostheses.¹⁸ 

This in vitro experiment rejected the null hypothesis, revealing differences in the total amount of biofilm formation between tested materials. The PTFE tape and gutta-percha presented lower biofilm colonization than cotton. There was no significant difference between gutta-percha and PTFE tape groups.

Abbreviations

BHI:

brain heart infusion

MTT:

methyl tetrazolium

OD:

optical density

PBS:

phosphate-buffered saline

PTFE:

polytetrafluoroethylene

The authors thank the collaborators in this study, carried out at the School of Dentistry of Rio de Janeiro State University, Rio de Janeiro, Brazil, and Luciana de Souza Velasque, an independent statistician, who reviewed the methodology.