Sealing agent reduces formation of single and dual-species biofilms of Candida albicans and Enterococcus faecalis on screw joints at the abutment/implant interface

The aim of this research was to evaluate the efficacy of a commercial sealing agent at the abutment/implant interface against microleakage of single and dual-species biofilms of Candida albicans and Enterococcus faecalis into external hexagon (EH) and Morse taper (MT) prosthetic connections. A total of 216 samples of implants and their abutments were tested. Six groups (n = 36) were evaluated based on biofilm and period of incubation (7 and 14 days). The implant connections EH and MT (n = 18) were divided according to the use of the material (n = 9) (EH-T and MT-T: with the sealing agent; EH-C and MT-C: control). The biofilms were analyzed by microbial counting (CFU/mL) and SEM analysis and photographs of the material in the screw joints were also taken. Data were analyzed by Student t test, two-way ANOVA and Bonferroni test. For the single-species biofilms, there was a significant reduction in the growth of E. faecalis when compared MT-C and MT-T or EH-C and EH-T at 7 and 14 days. The same was observed for C. albicans biofilms. For dual-species biofilms of E. faecalis and C. albicans, the sealing agent was more effective in preventing microbial infiltration into the MT connection at 14 days, while microbial infiltration did not occur into EH connections even in absence of the sealing agent for both periods of evaluation. Overall, these data suggest that the presence of the sealing agent reduces or eliminates the microleakage of E. faecalis and C. albicans biofilms into the implants regardless of the period of incubation.


Introduction
Numerous studies have reported high success rates with regard to dental implant treatment. However, several complications that may lead to implant loss can occur during and after the period of osseointegration. [1] One of the reasons for the failure of implants is the increased prevalence of peri-implant infections, which are multifactorial and immune-mediated a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 vibrations, by filling the voids between the threads, molding to the roughness, and forming a unique body [19].
Similarly, this study evaluated the sealing ability of a mono component, anaerobic, and thixotropic material with low resistance to disassembly, commonly being used industrially to adjust screws. This methacrylate-based product is suitable for locking and sealing threaded surfaces, the dismantling of which requires conventional hand tools. The product curing, when confined between metal surfaces in the absence of air, prevents loosening and leakage caused by impact and vibration. It is especially suitable for applications on less active surfaces, such as stainless steel and treated surfaces.
The aim of this research was to evaluate the efficacy of a sealing agent at the abutmentimplant interface by preventing infiltration and formation of single and dual-species biofilms of C. albicans and E. faecalis in two prosthetic dental implant connections (external and internal hexagonal Morse taper), at different incubation periods (7 and 14 days). The null hypothesis established was that there was no significant difference in the microbial infiltration and biofilm formation by C. albicans and E. faecalis when inoculated alone or together, regardless of the type of connection and the period of incubation.

Study groups and sealing agent
A total of 216 titanium alloys implants of 4 mm diameter were connected to their respective UCLA-type prosthetic abutments and were retained by the appropriate retaining screws, (DSP Biomedical, Campo Largo, Paraná, Brazil). Two types of implant connections, external hexagon (EH) (n = 108) and Morse taper (MT) (n = 108) were evaluated. Three types of biofilms were analyzed: C. albicans single biofilms (n = 36), E. faecalis (n = 36) single biofilms, and C. albicans and E. faecalis mixed biofilms (n = 36). For each type of biofilm, the sets were subdivided into two groups according to the use of the sealing agent: test groups ( 18]). The biofilms formed in these sets were evaluated for two time periods, 7 and 14 days (n = 9). For biofilm analysis, three experiments were carried out on independent occasions using three samples from each group and were performed in triplicates. (Fig 1)

Bacterial strains and growth conditions
Two microorganisms were used: C. albicans (ATCC 90028) and E. faecalis (ATCC 29212) [20]. The microorganisms were reactivated and cultivated separately in 5 mL Brain Heart Infusion (BHI) broth culture medium (Difco, Kansas City, MO, USA) to obtain the growth curve of the microorganisms, determined by their Optical Density (OD) values [21]. After reaching the log phase of growth (OD = 0.3 for C. albicans and OD = 0.5 for E. faecalis), microbial cultures were adjusted to 1.5 × 10 8 cells/mL, by dilution in the BHI broth.

Biofilm and collection assay for microbiological processing
Contamination control of the experiment. The experiments were carried out in a biosafety cabinet (Veco-Campinas-SP, Brazil). The sealing agent was sterilized by ultraviolet light from the cabinet for 20 minutes. Two calibrated operators performed the experiments. The first operator manipulated only the sterile materials with sterile gloves and scrubs. The second operator used conventional gloves and had contact with the rest of the instruments that were not sterile, thus avoiding the contact with sterile materials in order to maintain the aseptic conditions.
Bacterial contamination-Control group. The experiments were conducted with the first operator keeping the implant in a vertical position by means of hemostatic tweezers [22] and the second operator inserting 2 μL of sterile BHI broth culture medium into the implants. After that, the first operator positioned the respective abutments, threaded their bolts, and applied the torque (20 Ncm for MT implants and 30 Ncm for EH implants). Prosthetic abutments were closed with sterile cotton and temporary restorative material (Obtur-Maquira e Produtos Odontológicos S.A-Maringá-PR, Brazil) to prevent their opening and posterior infiltration into the implant.
Bacterial contamination-Test group. After 2 μL sterile BHI broth medium to be inserted into the implants, a thin layer of the sealing agent (previously sterilized with ultraviolet light for 20 minutes) was applied to the implant platform, to the abutment, and to the threads of the retaining screw, with a sterile micro-brush (Fig 2). Then, the respective abutments were positioned on the implants, their bolts were threaded, and the torque was applied. The same procedures adopted for the control group were conducted for the test group considering torque parameters and prosthetic abutments closure.   Specimens from both groups (test and control), were inserted into tubes containing 5 mL and 1.5 x 10 8 of either one of the bacterial cultures: E. faecalis, C. albicans, or a mixed culture containing both C. albicans and E. faecalis (2.5 mL of each microbial culture). The tubes were incubated at 37˚C in 5% CO 2 atmosphere for 7 and 14 days. Every 2 days, the culture medium was renewed to the same original volume in order to avoid microbial starvation.
Material collection and microbial counting. After 7 and 14 days of incubation, the culture medium inside the implants was collected. The specimens were removed from the tubes and rubbed in gauze soaked with 70% alcohol for external decontamination. The assembly was stabilized in an upright position and the prosthetic abutment screw was removed. Subsequently, 10 μL of saline solution (0.9% NaCl) was carefully inserted into implants and the walls of the implants were scraped with the aid of a 10μL micropipette tip. The solution was resuspended in order to recover the culture medium that had been previously placed and microorganisms that could be trapped between the threads [23]. Next, 5 μL of this solution was serially diluted, plated in three different media, according to the biofilm type and incubated at 37˚C in 5% CO 2 atmosphere for 48 hours. For single species biofilms, BHI agar media was used for total E. faecalis counting and Sabouraud Dextrose agar was used for total C. albicans counting. For dual-species biofilms, BHI agar containing amphotericin (20 µg/mL) was used for E. faecalis counting [24] and Sabouraud Dextrose Agar containing chloramphenicol (40 µg/mL) was used for C. albicans counting [25].

Biofilm analysis by scanning electron microscopy (SEM)
One representative specimen from each group (CM-C, CM-T, EH-C, EH-T after 7 and 14 days of single or dual-species biofilms) was analyzed by SEM. The abutment and implant were analyzed separately and the connecting region of both the pieces was also analyzed after positioning the specimen in a vertical position. The specimens were pre-fixed in serial dilutions of alcohol (70% for 5 minutes, 90% for 5 minutes, and 100% for 20 minutes), dried under aseptic conditions in a biosafety cabinet [24], and subjected to metallization with gold blasting. The analysis was performed at two distinct sites of the specimens, at 1000× magnification using a scanning electron microscope.

Visual analysis of bolted joints and sealing agent inside
The specimens of each test group were embedded in a colorless, self-curing acrylic resin (VIPI Flash, VIPI Produtos Odontológicos, Pirassununga-SP-Brazil) by means of an electric filler (Arotec PRS-30S -Arotec Indústria e Comércio-Cotia-SP, Brazil). They were then positioned and subjected to coronal cutting using a precision cutter (Isomet 5000, Buehler-Illinois, EUA). The parts obtained were photographed using a digital camera with a 100 mm macro lens (Canon EOS 70D -Ota-Toquio, Japan).

Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics version 17.1 (Statistical Package for the Social Sciences, IBM Corporation, New York, EUA). The Kolmogorov-Smirnov homogeneity test was applied to data and normal distribution was observed. In order to compare the mean CFU/mL count of the single-species biofilm assays of E. faecalis and C. albicans, the twoway ANOVA test was carried out followed by the Bonferroni test. A level of significance of 5% was considered statistically significant. For dual-species biofilms, student t-test was used. The influence of the sealing agent and the incubation period (7 and 14 days) were analyzed. The number of CFU/mL was logarithmically transformed into log (CFU + 1) due to the high microbial counts. The "+1" was added in order to factor in the zero values that were found in this study (log 1 = 0). Fig 3 shows the counting of E. faecalis or C. albicans recovered from implants after 7 and 14 days in single-species biofilms. Fig 3A shows total count (log (CFU/mL + 1)) of E. faecalis recovered from implants of control and test groups after 7 and 14 days of incubation. After 7 days of incubation, a statistically significant difference was observed in the growth of E. faecalis between the Morse taper control and test groups (MT-C vs. MT-T) (p = 0), with bacterial growth detected only in the control group (without sealing agent). The same performance was observed for external hexagonal connection (EH-C vs. EH-T), with bacterial growth observed only in the control group. Intergroup analysis showed a significant difference in the bacterial growth between the test groups of Morse taper connection at 7 and 14 days (MT-T 7 days vs. MT-T 14 days). After 14 days of incubation, the intragroup analysis showed a statistically significant difference (p = 0.04) in the bacterial growth in the conical connection groups (MT-C vs. MT-T), with the highest bacterial count in the MT-C group (5.22 ± 0.71). The hexagonal connection groups (EH-C vs. EH-T) showed a statistically significant difference in the bacterial growth in the two groups with growth detected only in the EH-C group (5.10 ± 0.83). For the control groups of both connections (MT-C vs. EH-C), there was no statistically significant difference in the bacterial growth, while the bacterial growth in the test groups (MT-T and EH-T) differed significantly from each other (p = 0.02), and the EH-T group did not present any bacterial growth at all.

Results
Fig 3B shows total count (log (CFU/mL + 1)) of C. albicans recovered from implants of control and test groups after 7 and 14 days of incubation. After 7 days of incubation, a statistically significant difference was observed in the growth of C. albicans between the test and control conical connection groups (MT-C vs. MT-T) as well as between the external hexagonal groups (p = 0) (EH-C vs. EH-T), with microbial detection only in the control groups (without sealing agent). However, there were no significant differences (p = 1) in the bacterial growth between the control and test groups (MT-C vs. EH-C and MT-T vs. EH-T). After 14 days of incubation, the intergroup analysis showed a statistically significant difference (p = 0) between the conical connection groups (MT-C and MT-T). On the analysis of the hexagonal connection group (EH-C and EH-T), a statistically significant difference (p = 0) was observed in the bacterial growth between groups, with growth observed only in the EH-C group (4.93 ± 0.09). Considering the intergroup analysis (MT-C vs. EH-C and MT-T vs. EH-T), the control and test groups did not present a statistically significant difference between themselves (p = 1). There was no statistical difference between similar connections as a function of time (p = 1).
Fig 4 shows total counts (log (CFU/mL + 1)) of E. faecalis and C. albicans in dual-species biofilms recovered from Morse taper implants connections, after 7 and 14 days of incubation.
No microbial growth was observed in the external hexagonal connections, irrespective of the microorganism and the incubation period. Thus, there was a statistically significant difference between the control and test group connections across periods of time regardless of the microorganism. After 7 days of incubation, significant difference was observed in E. faecalis counts in the MT-T group in comparison to its control (MT-C). After 14 days, there was no bacterial growth in the MT-T group, only in the MT-T group. Moreover, upon comparing the two periods of time, 7 and 14 days, there was a significant difference in the bacterial growth between the both groups MT-C and MT-T, across the time period. For C. albicans in the dualspecies biofilms, growth was detected only in the control group, regardless of the time of evaluation. We also observed that there was no significant difference in the bacterial growth between the control groups (MT-C 7 days and MT-C 14 days).  Fig 5F and 5G represent E. faecalis and C. albicans single-species biofilms, respectively, and it is not possible to observe biofilm formation in the internal region of the implant; however, surface irregularities indicate the presence of the sealing agent in this region. Fig 6H is a representative of dualspecies biofilm, which shows the same characteristics such as the surface irregularities in the absence of biofilm formation, as described above. Fig 6 shows representative micrographs of the EH specimens after 7 days of incubation with E. faecalis and C. albicans, in single or dual-species biofilms, obtained by SEM. Fig 6A  and 6E are representative of the EH-C and EH-T groups, respectively, where the outer portion in the region of the connection can be visualized from the images. Fig 6E shows the presence of surface irregularities on the hexagon, suggesting the presence of the sealing agent in this region, which cannot be observed in the control group (Fig 6A). Fig 6B, 6C and 6D, are representative of the EH-C group for biofilms of E. faecalis, C. albicans in single-species, and in dual-species biofilms; a great amount of extracellular matrix and biofilm are observed in these figures, while the Fig 6F, 6G and 6H, which are representative figs of the EH-T group, present only material particles with absence of the biofilm; Fig 6H shows   MT-T and EH-T (Figs 7E and 8E) in contrast with Figs 7A and 8A from MT-C and EH-C groups in which the presence of the sealing agent is not observed in the same region. The presence of spherical cell agglomerate, which is a phenotypic characteristic of E. faecalis, is observed in single-species biofilm of MT-C specimen (Fig 7B). Colonies characteristics of C. albicans were observed in single-species biofilm ( Fig 7C) and in dual-species biofilms (Fig 7D) of MT-C groups. Fig 7F shows some cells of E. faecalis in single-species biofilms. Fig 7G and 7H shows no microbial cells on the specimens of MT-T connections. Fig 8B and 8C shows single biofilms of E. faecalis and C. albicans. Dual-species biofilms of E. faecalis and C. albicans on EH-C connections was not detected in Fig 8D. The presence of microorganisms organized in biofilms is not observed on EH-T connections (Fig 8F, 8G and 8H); however, the sealing agent is identified on the entire internal surface of the implant, forming a homogeneous film on this surface. 9 and 10 show the longitudinally sectioned images obtained from the specimens with MT and EH connections, respectively. It was possible to visualize the interior of these connections, the relationship between the structures, and the presence of the sealing agent in the MT-T and EH-T groups. The red arrow indicates the presence of the sealing agent at the abutmentimplant interface, while the yellow arrow points to the sealant between the threads of the retaining screw and the internal thread of the implant at the uppermost portion of the screw joint. In the groups with MT-C and EH-T connections, it is possible to verify the absence of material at these interfaces.

Discussion
Single biofilms of E. faecalis and C. albicans infiltrated into the two connections (Morse taper and external hexagon) without any significant difference in the infiltration between either of However, when the microorganisms (E. faecalis and C. albicans) were inoculated together, evident difference was observed in the microbial infiltration between the external hexagon    connection groups in relation to the Morse taper connection groups. There was no microbial growth and infiltration into the EH implants, in either the control group or the test group, at 7 or 14 days. It is known that different geometries at the abutment-implant interface can interfere with the penetration of microorganisms into the implants [12]. This result differs from some literature reports [8,[26][27][28][29][30], which assert that the Morse cone type connection is superior to other connections with respect to the susceptibility to microbial infiltration.
However, in a study evaluating water infiltration associated with gentian violet-based dye at the abutment-implant interface with different connections [31], it was observed that the group that received Morse taper implants showed the highest water infiltration, while the groups that received external connection implants had a significantly lower water infiltration. Ricomini Filho (2010) evaluated the infiltration of Streptococcus sanguinis inside groups that received implants with external hexagonal connection and Morse taper, and were subjected to mechanical cycling. It was reported that in the Morse taper connection implant group, there was a 67% higher occurrence of bacterial infiltration in comparison to the groups of external hexagonal connection implants, regardless of whether they were subjected to mechanical cycling or not [32]. In the external hexagon group, the presence of bacteria was not observed in any part of the implant or its prosthetic component, which corroborates with the results found in the present study.
This might be due to the fact that the external hexagon on the implant connection platform may have acted as a physical barrier, which possibly prevented the interface microorganisms from penetrating the implant [32]. Furthermore, the fact that the present in-vitro study was conducted in a static manner may have made it difficult for the microorganisms to move towards the interior of the implant.
Another aspect to be considered is the difference in the value of the torque applied to the retaining screws of the abutments for HE and CM connections (30 Ncm and 20 Ncm, respectively). These values are, in general, used by several brands of implants. However, the study conducted by Silva-Neto (2012) evaluated the infiltration of high concentration of Escherichia coli (0.5 μL, 1.0 μL, and 1.5 μL) in external hexagon implants with screwed abutments using different torque values (10 Ncm, 20 Ncm and 32 Ncm). They observed that in the implants whose abutments were screwed with 32 Ncm torque, none of the specimens showed microbial infiltration. [33] This torque value is close to the value that was used in the present study for the HE groups, and may have been enough to maintain the ideal contact and bonding of the parts.
In contrast, the Morse taper implants showed infiltration and biofilm formation, indicating that even though the torque indicated by the manufacturer was applied, this may not have been enough to seal the interface. This result contradicts previous studies that have demonstrated the effectiveness of this connection in the containment of the microbial infiltration to its interface. [14,30,34] In case of mixed biofilms, microorganisms established interactions that may or may not favor their proliferation and metabolic activity. In dual-species biofilms showed in the present study, CFU counts were similar between E. faecalis and Candida albicans at 14 days, however, there was an increase in the E. faecalis counts comparing 7 and 14 days, showing bacterial growth. Using a nematode infection model, Garsin and Lorenz (2013) evaluated dual-species biofilms of C. albicans and E. faecalis and identified specific interactions occurring between these two pathogens that result in a potentially synergic relationship; a phenomenon in which microorganisms support each other's growth and proliferation, although a inhibition of hyphal morphogenesis of C. albicans was observed, attenuating its virulence and avoiding the worm killing (35). Cruz et al. (2013) showed, in a similar model host system for pathogenesis studies with different microbial species, these two important pathogens when together appear to inhibit each other's virulence: not only does E. faecalis attenuate killing by C. albicans, but a subsequent exposure to the fungus attenuates E. faecalis killing as well. They also demonstrated that the presence of C. albicans appeared to protect E. faecalis from cell death (36). Comparing 7 and 14 days of dual-species biofilms, there was a significant decrease in the CFU counts of E. faecalis (MT-T 7d x MT-T 14d), however, both microbial species did not show any growth inside the implants at this time of evaluation. We speculated that dual-species biofilms become thicker and denser outside the implant and the physical presence of the biofilm associated with the sealing agent was enough to completely block the infiltration. Gao et al. (2016) showed that E. faecalis was more resistant to starvation in coexistence with C. albicans, S. gordonii, A. viscosus, or L. acidophilus. The dual-species biofilm showed that E. faecalis formed thicker and denser biofilms on the root canal dentin and glass slides in coexistence with these species (37).
The same microorganisms evaluated in single biofilms presented a different behavior in relation to their ability to infiltrate the abutment-implant interface and to grow inside the implant, in comparison to the mixed inoculation assays. After 7 days of incubation, there was growth of the tested microorganisms only in the control groups (CM-C and HE-C for C. albicans and E. faecalis), while the test groups (CM-T and HE-T for C. albicans and CM-T and HE-T for E. faecalis) remained free of the formation of biofilms inside the implants. Thus, the effectiveness of the sealing material in preventing microbial infiltration of E. faecalis and C. albicans biofilms to abutment-implant interface sealing was evident post 7 days. This result negates the second hypothesis in this study that there is no significant statistical difference in microbial infiltration and formation of biofilms between the test and control groups, regardless of the connection type and the period of incubation.
After 14 days of incubation, microbial infiltration of E. faecalis to the CM-T group was statistically significant different from its infiltration into the CM-C group. CM-T group was positive for E. faecalis growth, however, on a smaller scale in comparison to CM-C (CM-T: 5.45 ± 0.49 and CM-C: 0.98 ± 1.95). Thus, there was an effect of the material in containing E. faecalis infiltration, however, to a lesser extent when compared to the HE-T group. This is justified by what has been already discussed earlier in relation to the different designs and properties of each connection. Thus, it is evident that the efficacy of the material at the abutmentimplant interface was important regardless of the incubation period for E. faecalis. However, no growth of C. albicans was observed in test groups that received the sealing material, regardless of the type of connection, even after incubating for 14 days, suggesting that the sealing material there was more effective against C. albicans compared to E. faecalis.
Jansen and Conrads (1997) have shown that the gap size at the abutment-implant interface varies according to the type of connection and the structural characteristics of the prosthetic abutment. In addition, they found that the mean vertical distance between the components is approximately 0 to 10 μm [35,36] and the horizontal distance is 60 μm [37,38]. However, the lack of formation of biofilm in the test groups by C. albicans is not surprising due to the morphology of the fungus, as C. albicans are physically large (4 to 6 μm) [39,40] with hyphae and pseudohyphae, which may have limited their mobility through the gaps into the implants of the test groups, since, in addition to the bonding of the pieces of the screw joint, there was the sealing agent present that may have prevented the entry of the microorganisms inside the implant. [39][40][41] This fact can be verified by the photographs (Figs 9 and 10) that show that the sealing agent occupies an important space at the abutment-implant interface and at the screw junction, which acts as a physical barrier, preventing the entry of larger microorganisms such as C. albicans. The photographic images corroborate with SEM images, which present biofilms in the outermost region of the implant, although growth (CFU/mL) was not observed in the portion that was collected from the implant. Thus, even if gap formation between implant and abutment is unavoidable [20], it is possible to prevent microorganisms from entering the implant chamber, preventing peri-implant diseases.
Upon analysis of the isolated biofilms, a significant difference was observed between the CM-T groups after 7 and 14 days of incubation, with microbial growth of E. faecalis observed only at 14 days for single-species biofilms. There was also a statistically significant difference between these two groups for mixed cultures of E. faecalis and C. albicans, with microbial growth observed only at 7 days. This might be due to the greater thickness of the biofilms formed by mixed culture of E. faecalis and C. albicans at 14 days because of which the microorganisms could not cross the abutment-implant interface and penetrate the implant in the presence of the sealing agent, unlike what occurred at 14 days in the isolated cultures of E. faecalis.
It is also important to note that one of the main advantages of the sealing agent used is its reversibility with screwed prostheses, which makes it possible to loosen the prosthetic part for modifications, repairs, adjustments, and maintenance of screwed prostheses. [19,42] A limitation of the study that should be noted is that the study was performed under static conditions, which do not mimic what actually occurs in the oral cavity with variations in temperature and forces that directly and indirectly affect the abutment-implant assembly.
Considering these results and their variability in microbial infiltration depending on isolated or mixed biofilms, it was evident that the use of the sealing agent was effective in containing the microbial infiltration of the tested microorganisms. In this context, important studies are being carried out to test microbial infiltration through connection implants by other microorganisms and their combinations and to perform mechanical analysis, evaluate the properties of this material in relation to its cellular biocompatibility, and to evaluate the tissue response in order to verify the reactions of the organism when in contact with this product, before recommending it for clinical use.

Conclusion
Based on the results obtained and after considering the limitations of this study, it is possible to conclude that the presence of the sealing agent reduces or eliminate the infiltration of biofilms of E. faecalis and C. albicans into the implants, irrespective of the time period of incubation. The external hexagonal connection was more effective than the Morse Taper connection against microbial infiltration for dual-species biofilms, regardless of the time after microbial incubation and the use of the sealing agent.