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Basic and Clinical Research

Effect of 5 Popular Disinfection Methods on Microflora of Laboratory

Customized Implant Abutments

Homayouni, Ali DDS*; Bahador, Abbas PhD; Moharrami, Mohammad DDS; Pourhajibagher, Maryam PhD§; Rasouli-Ghahroudi, Amir Alireza DDS, MSc; Alikhasi, Marzieh DDS, MSc

Author Information
doi: 10.1097/ID.0000000000000906
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Abstract

Contemporary dentistry considers dental implants as a successful replacement option for lost teeth because it is estimated that implant supported single crowns have a survival rate of 96% after 5 years of function.1 Marginal bone loss (MBL) has been of paramount importance in dental implant success rate assessment2; so, it has been always of great concern to preserve every tenth of a millimeter of alveolar bone around dental implants. Many efforts have been made to reduce the MBL around dental implants by introducing new procedural concepts or implant design,3–5 among which some were perceptible, including implant type (1 piece vs 2 piece),6 abutment type (platform switched vs matching platform),7 level of implant-abutment junction (bone level vs tissue level),8 and time of definitive abutment placement (at the time of implant placement vs after repetitive disconnection and reconnection of abutment).9

Early MBL around submerged implants occurs before implant-abutment connection, which seems to be inevitable up to the present.10 A second phase of MBL occurs after uncovering submerged implants until the delivery of final prosthesis, and finally, the last, but continuous phase of MBL occurs after loading of implants.11 Proper soft tissue integration at the transmucosal part of a dental implant is a prerequisite to seal the adjacent alveolar bone from the oral environment.12,13 Soft tissue attachment, as a barrier to oral cavity, has a key role in preserving marginal bone height during the second phase and last phase of MBL. It is important to keep in mind that the quality of soft tissue attachment is influenced by the properties of the implant components that are in contact with the soft tissue.14

During the fabrication procedure of final prosthesis, implant abutments are sent to dental laboratory and sent back to dental office repeatedly. Hence, implant abutments become contaminated with various microorganisms and debris.15,16 Microorganisms and their endotoxins can cause osteoclastogenesis and subsequent bone resorption, specifically when there is a microgap due to debris that remained at the implant-abutment connection.17 Besides microgap formation, debris, such as titanium microparticles produced during abutment customization, can act as a foreign body and produce inflammatory response, which may influence soft tissue healing around implants.18

So, it is preferable to decontaminate abutments sent from dental laboratories before abutment connection to the implant. It has also been shown that decontamination of implant abutments may result in better soft tissue attachment and maintenance of marginal bone around implants19,20 because application of a decontamination method can have an impact on different features of titanium surfaces. Higher surface roughness facilitates accumulation of bacterial biofilm.21,22 It has been also shown that epithelial cells and fibroblasts adhere to machined titanium surfaces with higher strength when compared to rough surfaces.23,24 Several studies have evaluated the effect of surface roughness on soft tissue integration, at microscale; however, there is a lack of evidence regarding importance of nanoroughness in soft tissue integration. The key point in titanium abutment decontamination seems to be the preservation, at least, or improvement of chemical and topographic characteristics of titanium surface while reducing or eliminating microorganisms at the same time.

Currently, there is a wide heterogeneity among clinical centers worldwide in treating customized abutments before abutment connection to implant.25 The aim of this study was to assess antimicrobial efficacy of 5 different decontamination procedures as clinically feasible alternatives, and to show the possible effects of these procedures on titanium surface roughness.

Methods and Materials

Microbial Analysis

After a careful design of an in vitro study to assess the effect of 5 decontamination methods and their effect on microbial load and surface roughness of titanium discs, this study was approved by the research committee of Tehran University of Medical Sciences with the ethical approval number IR.TUMS.REC.1394.757. Microbial species were selected based on a previous study on microbial contamination evaluation of customized titanium abutments.16 We used a culture of reference stock of Micrococcus luteus American Type Culture Collection (ATCC) 10240, Acinetobacter baumannii ATCC 19606, Enterococcus faecalis ATCC 29212, Candida albicans ATCC 10231, and Bacillus subtilis ATCC 6633 cultivated on brain-heart infusion broth (Merck, Darmstadt, Germany). The microbial species were categorized into 2 groups, spore-forming (B. subtilis) and non–spore-forming (M. luteus, A. baumannii, E. faecalis, and C. albicans). These 2 groups were cultivated separately on 2 groups of 18 machined titanium discs (2.5 × 10 mm, Dentium Co., Seoul, Korea).

Suspensions of M. luteus, A. baumannii, E. faecalis, and C. albicans were provided separately at logarithmic phase, corresponding to optical density at a wavelength of 600 nm (OD600) of 0.3, 0.5, 0.4, and 0.08, respectively. One milliliter of each suspension was mixed together and incubated for 48 hours at 37°C, and a multispecies biofilm of 3.2 × 106 (SD: 1.6 × 105), 1.4 × 106 (SD: 1.9 × 105), 1.5 × 106 (SD: 6.5 × 105), and 1 × 105 (SD: 1.7 × 104) colony-forming unit (CFU)/mL/disc for each microorganism was eventuated, respectively. To make a B. subtilis biofilm with CFU/mL/disc of 3.5 × 106 (SD: 1.1 × 105), every disc was suspended in 1 mL of a suspension of B. subtilis with OD600 of 0.2 and incubated for 48 hours at 37°C to form spores. Each group of discs was divided into 6 subgroups to be subjected to different decontamination procedures (Table 1). Laser decontamination was performed using GaAlAs and Er:YAG lasers (Figs. 1 and 2).

Table 1
Table 1:
Decontamination Subgroups of Microbial Assay
Fig. 1
Fig. 1:
GaAlAs laser irradiation on titanium disc. GaAlAs laser was irradiated on the whole surface of discs held by pliers in a noncontact continuous wave mode. Second sterile pliers were used to hold the disc, making it possible to irradiate the area covered by the first pliers.
Fig. 2
Fig. 2:
Er:YAG laser irradiation on titanium disc. Er:YAG laser was irradiated on the whole surface of discs held by pliers in a noncontact pulsed wave mode with 50/50 air-water spray. Second sterile pliers were used to hold the disc, making it possible to irradiate the area covered by the first pliers.

To measure remnants of microbial contamination after each decontamination procedure, discs were transferred into 1.5-mL microtubes containing 1-mL phosphate buffered saline. Adherent bacteria on the discs were dislodged by ultrasonication for 5 minutes in a 150-W ultrasonic bath (Branson Ultrasonics Co., Shanghai, China) operating at a frequency of 50 Hz. Ultrasonication was followed by rapid vortex mixing (Kiagen, Tehran, Iran) at maximum power for 1 minute. Microbial counting was determined using Miles and Misra method26; briefly, decimal serial dilutions of each suspension were made up to 10−4; then, 10 μL of 10−1, 10−2, 10−3, and 10−4 suspension was plated on selective agar plates corresponding to each microorganism (Table 2) and incubated at 37°C under aerobic condition. After 24 hours, colonies grown on selective agar plates were counted as CFU/mL/disc.

Table 2
Table 2:
Selective Culture Media Corresponding to Each Microorganism Were Used to Detect The Residual Contamination by Microorganisms

Surface Roughness Analysis

Investigation of the effect of each decontamination procedure on surface roughness was performed using 23 sterile machined titanium discs. The discs were divided into 1 control group of 5 discs and 9 test groups of 2 discs (Table 3).

Table 3
Table 3:
To Detect Any Change in Surface Roughness of Titanium Discs Due to Decontamination Methods, the Effect of Nine Different Subgroups Were Investigated

After treatment, each titanium disc surface was scanned using an atomic force microscope (AFM) (Nanosurf Mobile-S, Liestal, Switzerland) at 6 random spots (3 spots on each side), and the dimension of each spot was 9.21 × 9.24 µm2. Six surface roughness parameters were selected based on previous studies,27,28 including 4 amplitudinal parameters (Sa, Sq, Ssk, and Sku), 1 spatial parameter (Sal), and 1 hybrid parameter (Sdr). A short description of each parameter is shown (Table 4). Parameters were calculated using Mountains Map Universal software, version 7.3.7904. Readers are referred to an article by Gadelmawla et al29 for more information about roughness parameters (Fig. 3).

Table 4
Table 4:
Among the Numerous Surface Roughness Parameters, Six Common but Comprehensive Parameters Were Chosen to Describe the Surface Roughness Alterations
Fig. 3
Fig. 3:
Image of a scanned point of a titanium disc by AFM rendered by Mountains Map Universal software. Roughness parameters were calculated on 6 different points (an area of approximately 100 μm2) of each disc.

Data Analysis

The Kolmogorov-Smirnov test was applied to verify the normality of collected data. The comparison of groups based on decontamination method, species, and their interactions was analyzed using two-way analysis of variance (ANOVA). Tukey honestly significant difference and t-tests were used for pairwise comparison among groups. The effect of decontamination methods on each surface roughness parameter was analyzed using one-way ANOVA. A probability level (P-value) of less than 0.05 was considered to indicate statistical significance. All statistical analyses were performed using SPSS software (version 23.0).

Results

Microbial Analysis

All decontamination groups could reduce microbial load significantly. Er:YAG, high-pressure steam, H2O2, and NaOCl groups could completely eliminate all the cultured microorganisms; however, GaAlAs could not eliminate microorganisms completely. Descriptive results of microbial count after decontamination by diode laser are demonstrated. (Table 5). C. albicans was the only microorganism that was eliminated by all the decontamination procedures. Among resistant microorganisms, M. luteus and A. baumannii were the most resistant species. Two-way ANOVA showed that different decontamination methods and various species had significant effect on reducing microbial count (P < 0.001). To assess pairwise differences among bacterial species, a post hoc statistical test was performed (Table 6).

Table 5
Table 5:
Except for C. albicans, Other Microorganisms Still Could be Detected on Titanium Discs After Decontamination by GaAlAs Laser
Table 6
Table 6:
Microbial Species Showed Different Levels of Resistance to Decontamination by GaAlAs Laser

Surface Roughness Analysis

Surface roughness measures of different groups are depicted in Figure 4. According to the one-way ANOVA statistical test, the surface roughness alteration was statistically significant for Sa, Sq, and Sdr parameters (Table 6). The Tukey test was performed to assess pairwise differences among decontamination groups for each parameter (Table 7). Sa and Sq parameters were subjected to statistically significant increase only in the diluted NaOCl group, whereas Sdr was increased significantly in both diluted and concentrated NaOCl groups.

Fig. 4
Fig. 4:
Descriptive results of surface roughness analysis. Sa (A), Sq (B), Ssk (C), Sku (D), Sal (E), and Sdr (F). There was no significant difference between the treated and nontreated discs, except for discs treated by diluted NaOCl according to Sa, Sq, and Sdr parameters and discs treated by absolute NaOCl according to Sdr parameter.
Table 7
Table 7:
Surface roughness of titanium disks were evaluated on treated and non-treated disks

Discussion

Up to the present, among the variety of different disinfectants, antimicrobial efficacy of plasma of argon and ultrasonic bath containing chemical decontaminants have been compared with high-pressure steam, which is commonly used in dental laboratories. Although both plasma of argon and ultrasonic cleaning could eliminate all the present microorganisms, unlike the high-pressure steam, these techniques have their own drawbacks.15 Ultrasonic cleaning is time consuming; moreover, immersion of several abutments in a single ultrasonic bath may cause difficulty in finding the original site and direction of each abutment after decontamination. Despite the excellent antimicrobial and surface activation effect of plasma of argon, the device is not routinely present in dental clinics or laboratories. Hence, it seems necessary to evaluate other techniques that are more accessible and routinely used, regarding their effects on microorganisms and titanium surface characteristics. Chemical disinfectants such as sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2) are relatively common and easy-to-use disinfectants in dental clinics. However, laser as a novel technology is being widely used by clinicians, and it is present in many clinics nowadays; besides, by laser irradiation, it is possible to disinfect an abutment in 1 or 2 minutes without those difficulties mentioned about immersion techniques.

To the best of the author's knowledge, this is the first study that compares the antimicrobial efficacy of 5 of the most trending methods (NaOCl, H2O2, high-pressure steam, GaAlAs laser, and Er:YAG laser) for decontamination of titanium discs contaminated by microbial species that were found on titanium abutments received from dental laboratories. Except GaAlAs laser, all aforementioned methods could successfully sterilize titanium discs resembling the gingival portion of titanium abutments. GaAlAs laser, like the other groups, could eliminate C. albicans totally but failed to eliminate A. baumannii, M. luteus, E. faecalis, and B. subtilis completely.

C. albicans and E. faecalis have been detected in implant-abutment connection and periimplant sulcus of implants affected by periimplantitis30,31; so, the authors believe that implant-abutment connection site can be a potential microbial reservoir predisposing patients to periimplantitis. Coculture of C. albicans with other microorganisms anaerobically results in increased hyphal development, expression of genes involved in adhesion and tissue damage32; therefore, presence of C. albicans in anaerobic conditions such as in the gap between internal surfaces of implant components is of paramount importance and should be taken into consideration. In addition to C. albicans and E. faecalis, A. baumannii has been also more frequently detected in subgingival biofilm of seropositive HIV patients with periodontitis than healthy patients.33,34 Although M. luteus and B. subtilis are two of the most common microorganisms found on customized abutments received from dental laboratories,16 to the best of the knowledge of the authors, no relationship have been found yet, between them and periodontitis or periimplantitis since present. However, hypothetically, these microorganisms might play a role in biofilm formation that could make other pathogenic microorganisms more resistant to the antimicrobial agents.

Canullo et al19 demonstrated that in periodontally healthy patients, decontamination of implant abutments by plasma of argon before insertion in the mouth results in decrease of MBL around dental implants. Similar results were obtained in patients with history of periodontitis after 5 years of follow-up.35 Moreover, from a histologic point of view, it has been demonstrated that treatment of sterile titanium abutments with plasma of argon resulted in increased density of more oblique collagen fibers in periimplant mucosa36 and could accelerate proliferation of fibroblasts and their initial adhesion to titanium surface.37 Besides these findings, it is not clear yet whether beneficial effects of decontamination of abutments results from elimination of contaminants or an improvement in surface features of titanium that may occur after plasma treatment. It has been shown that as the wettability of the surface increases, the number and attachment tightness of the cells rises, which improves connective tissue healing.38,39 Plasma treatment increases titanium surface wettability,40 but this is not unique to plasma treatment; there are other decontaminants with relatively similar effects on titanium surface. Ayobian-Markazi et al41 showed that treatment of sandblasted acid etched titanium surface using Er:YAG laser with settings similar to our study increases the wettability of the surface. A similar effect was shown for NaOCl42 and H2O243 treatments, which enhances epithelial cell growth on machined titanium surface.43

Inconsistent with our results, Gosau et al44 demonstrated that although NaOCl (1%) and H2O2 (3%) had antimicrobial effect on machined titanium discs contaminated by oral biofilms, none of the disinfectants could sterilize titanium discs. This contrast might be addressed by considering the more complex biofilm formed on the discs in oral cavity.

Several studies have been conducted evaluating bactericidal efficacy of different types of lasers.45–47 Because these studies have considerable differences regarding laser settings, surface roughness of titanium, and microbial composition of biofilm, it is not possible to compare their results. For instance, in this study, C. albicans was the only species that could be eliminated completely in both laser groups. Inconsistent with our results, Sennhenn-Kirchner failed to achieve complete elimination of fungal biofilm on machined titanium surface, although the laser settings used in their study were relatively similar to our laser settings (Er:YAG; 2940 nm, 100 mJ, 10 Hz, 300-μm pulsed mode applied for 80 seconds) (diode laser; 810 nm, 1 W, continuous wave mode applied for 20 seconds with 4 repetitions after 30-second pauses each). This failure might be related to formation of a mature C. albicans biofilm in the absence of other microorganisms, which is not realistic in periimplant environment.

It is important to take the limitations of this study into consideration when interpreting the findings; first, the samples (titanium discs) were simplified models of titanium abutment. Abutments have a more complex structure especially in the plaque-retentive threaded portion that might complicate decontamination procedure; second, other contaminants such as debris remained from prosthesis fabrication on abutments or titanium particles created during abutment customization accompanied by lubricant15 were absent in this study design. These chemical contaminants might compromise microbial decontamination efficacy; third, only 5 of 49 species identified on contaminated abutments, which were more prevalent or resistant, were selected based on the previous study and entered into study design.16 These drawbacks might justify different results of a previous study conducted by Canullo et al,15 in which high-pressure steam with the same pressure as that in our study could not eliminate all the microbial contaminants present on abutments after customization.

In this study, C. albicans was the only species that was eliminated by the GaAlAs laser completely, despite significant reduction of other species in number. Probably more powerful laser settings should be used to eliminate residual bacteria; however, it is important to consider probable adverse effects of higher power laser settings. GaAlAs laser has an intensive thermal effect, although it has been shown that there is no critical change in titanium surface irradiated by power of 3 W, when evaluated under scanning electron microscope48; besides probable negative effect on titanium surface, thermal effect could harm surrounding bone of an implant when the laser is used intraorally. Other factors also should be taken into account to detect possible harmful effects of laser treatment. For instance, in the abovementioned study, it was demonstrated that Er:YAG could create cracks on machined titanium surface by 500 mJ energy in each pulse with a frequency of 10 Hz.48

It has been demonstrated that increase in surface roughness, both in microscale and nanoscale, increases the wettability and adhesive force of the attached fibroblasts to titanium surface, whereas the proliferation rate of cells on the surface will be decreased.49 To the best of the authors' knowledge, there is a lack of evidence in the literature regarding the influence of decontamination protocols on surface roughness. Different concentrations of chemical solutions and different settings of lasers have been used in the literature to decontaminate titanium surfaces. To see how the concentration of chemical agents and more powerful settings of laser irradiation affects the surface roughness of titanium, 10% NaOCl and 35% H2O2 solutions, and GaAlAs laser (810 nm, CW, 3 W, 400-μm fiber, 1-mm distance, 1 minute) and Er:YAG laser (2940 nm, pulse mode, 500 mJ, 10 Hz, 230-μm noncontact handpiece, 4-mm distance, 50/50% air-water, 1 minute) were tested on sterile titanium discs besides the experimental groups that were used for the decontamination in this study. Among the test groups, surface roughness was increased significantly at nanoscale only in the NaOCl group. Our results regarding 3% H2O2 group is consistent with a previous study in which 3% H2O2 did not change titanium surface roughness significantly.43 In a study conducted by Stubinger et al, GaAlAs laser with power of 1 and 3 W did not cause significant change in Sa parameter of polished titanium surface. Inconsistent with our results, Er:YAG laser with 500 mJ/10 Hz energy/pulse could change the surface roughness significantly; however, lower power settings did not change the surface roughness.48 This contrast can be contributed to different methods of roughness measurement. They used confocal white light microscope (CWLM) to scan an area of 770 × 798 µm2 with a lateral resolution of 1.5

, whereas we used AFM to scan an area of 9.21 × 9.24 µm2 with a subnanometer lateral resolution. Although CWLM scans a wider target area, AFM is a more accurate tool to measure surface roughness.

Besides the effect of surface roughness on soft tissue healing, surface roughness could potentially affect microbial accumulation. It has been stated that there is a threshold roughness of 0.2 µm regarding microbial plaque accumulation, below which (Ra < 0.2 µm) there is no difference regarding microbial accumulation and composition. In a recent study, Webb et al demonstrated different behavior and accumulation tendency of Staphylococcus aureus on subnanometrically smooth titanium surfaces with different values of surface roughness parameters. It seems necessary to reevaluate microbial behavior and accumulation regarding different parameters rather than the single traditional parameter of Ra.

Because Er:YAG laser with the settings used in this study can decontaminate polished titanium surface completely with no change in surface roughness, the authors believe that its application may be useful to decontaminate abutments and/or fixtures in the clinical situations in which decontamination must be done intraorally. Other methods could be useful before abutment installation in the mouth; however, the clinical outcome of different decontamination methods of titanium abutments, regarding their influence on the degree of MBL around implants, implant success rate, and incidence of periimplantitis should be evaluated. Moreover, assessment of chemical contaminants' removal efficacy of the abovementioned methods and their influence on surface element composition of titanium seems logical, because surface element composition can affect cellular behavior. It is also recommended that in future studies, different laser settings be tested, not only by changing the power, but also by changing other parameters such as mode of irradiation (pulse/continuous), pulse width, irradiation time etc. Finally, it is important to note that these results cannot be extrapolated to titanium surfaces other than machined titanium surface. Rough titanium surfaces, additive or subtractive, definitely have different behavior regarding plaque accumulation, ease of decontamination, and surface alterations during decontamination procedures.

Conclusion

Within the limitations of this study, all the methods could decontaminate machined titanium surfaces, although complete microbial elimination was achieved in sodium hypochlorite (NaOCl), hydrogen peroxide (H2O2) high-pressure steam, and erbium-doped yttrium aluminium garnet (Er:YAG) laser groups. None of the methods altered surface roughness significantly, except for NaOCl. Further investigations, especially randomized clinical trials, should be conducted to evaluate the potential benefits or adverse effects of each method.

Disclosure

The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article.

Approval

This study was approved by research committee of Tehran University of Medical Sciences with the ethical approval number IR.TUMS.REC.1394.757.

Roles/Contributions by Authors

A. Homayouni: designed the whole study, conducted the laboratory tests, collected data, and wrote the manuscript. A. Bahador: designed microbiologic part of the study, interpreted microbiologic tests results, and edited the manuscript. M. Moharrami: conducted AFM tests, interpreted AFM tests results, and drafted the manuscript. M. Pourhajibagher: designed microbiologic part of study, conducted the microbiologic tests and interpreted the results, and edited the manuscript. A. A. Rasouli-Ghahroudi: designed the study, conducted the laboratory tests, and edited the manuscript. M. Alikhasi: designed the whole study, conducted laser tests, conducted statistics, edited the manuscript, and provided budget support.

Acknowledgments

The authors thank Dr. Reza Fekrazad and Dr. Nasim Chiniforoush for their contribution in the laser setup and reviewing the manuscript.

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Keywords:

periimplantitis; roughness; laser

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