|Year : 2011 | Volume
| Issue : 2 | Page : 51-57
Evaluation of the bacterial leakage along the implant-abutment interface
Renata Faria1, Liliana Gressler May1, Diego Klee de Vasconcellos2, Cláudia Ângela Maziero Volpato2, Marco Antonio Bottino1
1 Department of Dental Materials and Prosthodontics, São Paulo State University-UNESP, São Paulo, Brazil
2 Department of Dentistry, Federal University of Santa Catarina, Florianópolis, Brazil
|Date of Web Publication||30-Dec-2011|
Av. Conselheiro Nebias, 628/Cj 35, Santos, São Paulo, CEP: 11045-002
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context : Recent studies observed contamination of the inner parts of dental implants through bacterial penetration along the implant-abutment interface that may cause malodor and inflammation of peri-implant tissues.
Aims : Evaluate in vitro bacterial leakage along the implant-abutment interface, comparing three types of connections : EH, IH and MT.
Materials and Methods : Under sterile conditions, a colony of E. coli was inoculated in the apical portion of abutment screws, which were fixed to implants with a torque of 20 Ncm. Samples with immediate external contamination were discarded, while remaining specimens were placed in test tubes containing TSB. The broths that showed turbidity within the seven-day study period were planted in Petri dishes with TSA, and incubated in a bacteriological stove at 37°C for 24 h. At the end of the evaluation period, all assemblies were separated, and the internal content was collected using absorbent paper cones and saline water, and again planted to assess bacterial viability. The samples that did not contain viable E. coli were dismissed from the final results. As a result, 38 samples with EH, 40 with IH and 41 with MT connections were evaluated.
Statistical Analysis Used : Survival curves were analysed using the Kaplan-Meyer test and compared by log-rank statistics.
Results : There were no differences between the EH (10.53%), IH (4.88%) and MT (7.50%) connections.
Conclusions : Bacterial infiltration occurred similarly in all three types of connections between abutments and implants, despite the different configurations of the interface.
Keywords: Microbiology, prosthodontics, soft tissue-implant interactions
|How to cite this article:|
Faria R, May LG, de Vasconcellos DK, Maziero Volpato CÂ, Bottino MA. Evaluation of the bacterial leakage along the implant-abutment interface. J Dent Implant 2011;1:51-7
|How to cite this URL:|
Faria R, May LG, de Vasconcellos DK, Maziero Volpato CÂ, Bottino MA. Evaluation of the bacterial leakage along the implant-abutment interface. J Dent Implant [serial online] 2011 [cited 2020 Sep 22];1:51-7. Available from: http://www.jdionline.org/text.asp?2011/1/2/51/91280
| Introduction|| |
Despite the excellent success rates in osseointegrated implant rehabilitation, many flaws have been described related to surgical techniques and mechanical microbiological factors. ,, Bacteria and their products may cause inflammatory reactions in the peri-implant soft tissue, ,,,, which is why the important role of microorganisms in implant survival should be considered.
Longitudinal studies have demonstrated that implants have good longevity, verifying that the annual average bone loss is 1.5 mm in the first year and decreasing to 0.2 mm per year in the following years. ,,,, These criteria for success are related to consistent observations of the external hexagonal implant systems and prosthetic connections. Additionally, the implant-abutment interface is placed at the crest of the alveolar bone. , Implants with bone loss can remain in function, however, clinical sequelae of bone loss usually affects the final esthetic results of the oral rehabilitation. The peri-implant bone loss can lead to proportional gingival recession, , resulting in a lower height of the papilla due to the increased distance between the contact point of the teeth and the bone crest. 
In the conventional technique, the implant is placed at the bone crest level and, after a period of 3 to 6 months, a prosthetic abutment is installed on the implant to connect the implant to future prosthetic restorations (crowns, bridges or dentures), creating a micro-gap between the implant-abutment interface that could present a risk for bacterial colonization. ,, Broggini et al.  found infiltration of inflammatory cells (predominantly white blood cells) around the implants, which vary according to the implant design. A histological analysis  of failed implants identified a high-grade bacterial colonization at the level of the implant-abutment interface, legitimizing the hypothesis that micro-gaps at that level could exhibit bone loss. Such micro-gaps can act as a bacterial niche, favoring the loss of the peri-implant mucosal seal, followed by the alteration of the clinical and microbiological parameters of tissues, promoting periodontal disease and compromising osseointegration. ,,,,,,,,
Several attempts to obtain a more secure connection between the abutment base and the implant fixture have been studied. External and internal connections, such as hexagonal, conical (Morse taper) or a combination of both, are generally the most commonly used connections. Conical connections appear to be mechanically more stable than the external hexagonal, , due to the precision of fit between the implant and the abutment interface, preventing the passage and deposition of bacteria. 
In vitro,,,,,, and in vivo studies ,,, have assessed bacterial infiltration into the interfaces between abutments and implants with various types of prosthetic configurations; however, controversial results were obtained. Micro-gaps between the components is inevitable, ,, but their clinical significance has been underestimated by manufacturers and clinicians. Considering that bacterial infiltration is one of the main parameters in determining the quality of the connections, the objective of this current study is to evaluate different types of prosthetic connections to assess bacterial infiltration through interfaces between the abutments and implants.
| Materials and Methods|| |
To assess bacterial infiltration with three types of prosthetic connections, 150 assemblies of abutments and implants (Conexão Sistemas de Prótese Ltda., Brazil) were used, and divided into three groups (n=50) [Table 1]. The Morse taper implant was used with two abutment connection systems, internal hexagon and Morse taper; therefore, G2 and G3 have the same type of implant, but different prosthetic connections. The inverted direction was used for testing bacterial infiltration along the implan-abutment, i.e., the passage of bacteria from the inside part of the implant to the external environment. Escherichia More Details coli (ATCC 25922) was cultured in plates containing Tryptic Soy Agar (TSA) (Acumedia Manufacturers, EEUU). The bacterial culture plates were incubated at 37 ± 1°C for 24 h to allow for bacterial growth. During the experimental stages, some care was taken to avoid any risk of contamination. All procedures were performed in a sterile environment, with sterile gloves (Embramac, Brazil) and a laminar flow chamber (Valiclean, Brazil), which was covered with sterile surgical cloth (Asséptico, Brazil). All equipment was previously steam sterilized in an autoclave (Baumer, Brazil) at 121° for 15 min. The experiment was performed by three operators who performed the same functions until the end of the experiment. Two operators worked within the laminar flow chamber and the other aided providing material and recording the information.
Each implant was held in a vise (JCM, Brazil) using sterile tweezers (Golgran, Brazil), to allow work to be performed on it. Using a platinum needle (Cial, Brazil) flamed in a Bunsen burner (Orca, Brazil), a single colony of E. coli was picked and immediately inoculated on the apical portion of the abutment screw. After inoculation, the prosthetic abutments were connected to implants using a torque of 20 Ncm (Reference torque wrench, Conexão Sistemas de Prótese Ltda., Brazil).
To verify immediate external contamination, each assembly, implant-abutment, was passed on the Petri dish More Details with TSA, covered with solubilized agar, and removed from the plate. Subsequently, each assembly was placed in a test tube (Alfa Products for Hospitals and Laboratories, Brazil) containing 4 mL of Tryptic Soy Broth (TSB) (Acumedia Manufacturers, USA). Plates containing solid agar were incubated at 37°C for 24 h to check if there was any bacterial growth. The assemblies in contaminated plates were discarded from the study.
G1 and G2 samples were suspended and stabilized in a test tube, using a chromium-nickel orthodontic wire with a 0.30 mm diameter (Dental Morelli, Brazil) to maintain the interface region between the abutment and the implant that stayed in contact with the culture medium, without the possibility of passage of bacteria from the interface between the abutment and the abutment screw. G3 samples did not require this procedure because they are a one-piece solid abutment and there was no other way for the bacteria to exit from the inner portion of the implant, except at the interface between the abutment and the implant.
All of the test tubes were enumerated, placed in a test tube rack in a vertical position and incubated at 37°C. Daily monitoring of the tubes was conducted to verify the possible passage of bacteria from the inner part of the implant to the broth. Infiltration was indicated by turbidity of the culture medium. In the samples that showed turbidity, the broth was cultured on a TSA Petri plate. The plates were incubated at 37°C for 24 h to observe bacterial growth. To certify that E. coli was present, Gram staining was performed on both the turbid broth and the colonies on the agar. Staining and bacterial morphology were observed under light microscopy (Carl Zeiss, Switzerland).
Bacterial evaluation was conducted over a period of seven days. This period was determined based on a pilot study in which the implants were opened daily and the material was collected at the inner part using endodontic sterile paper cones (Tanari, Brazil) and saline water, verifying if viable bacterial occurred. After this period, the samples with no evidence of turbidity were removed, and their abutments unscrewed from the implants in a sterile environment (inside the laminar flow chamber). The contents were collected with sterile paper cones and sterile saline, and put into TSA Petri dishes. This procedure was performed to verify the viability of the bacteria and the sealing between abutment-implant interfaces. The inoculated plates were kept on a bacteriological stove (Callmex, Brazil) at 37°C for 24/48 h to verify bacterial growth. Samples with external contamination (turbid broth) and those that did not show turbidity, but did not present bacterial viability within seven days, were discarded from the study.
In addition to the analysis of bacterial infiltration, the interfaces between six assemblies of each type of prosthetic abutment and each type of implant system were evaluated using a digital optical microscope (Mitutoyo, model AT112-50F, Mitutoyo Corporation, Japan). For this evaluation, the assemblies were embedded in acrylic resin and longitudinally sectioned. The purpose of this analysis was to illustrate the mechanical connection between the abutment and the implant.
| Results|| |
The study consisted of 50 samples for each of the three models of prosthetic connections and their respective implant connections: Morse taper, external hexagon and internal hexagon, with a total of 150 assemblies (abutments and implants) were tested. Depending on the number of samples discarded, the quantity of samples considered for the calculation of the results was reduced [Table 2].
Of the total evaluated connections (119), 7.56% showed infiltration after seven days. Four samples (10.53%) presented bacterial infiltration through the implant- abutment interface in G1, two samples (4.88%) in G2 and three samples in G3 (7.5%).
The results were statistically analyzed using a multiple comparison test on proportions, which verified that an equal prevalence of infiltration occurred in the three types of prosthetic connections used in this present study. A Kaplan-Meier analysis was performed to compare survival curves (to compare the probability of infiltration at any seven days) of all the implant-abutment connections.
In G1, the first sample presented bacterial infiltration after three days of immersion; the second and third samples presented infiltration after five and seven days, respectively. In G2, bacterial infiltration was detected after five and six days; and in G3 after two, three and four days. The remaining samples of G3 presented bacterial infiltration by the end of the seven days.
The log-rank test was used to determine differences in survival curves between groups (χ2 =0.879; gl=2; P=0.644). The test indicated that all groups showed similar behavior with regards to bacterial infiltration as time passed, as shown in [Figure 1].
|Figure 1: Comparison of probability distribution curves of the different types of prosthetic connections with regard to bacterial infiltration as time passes by|
Click here to view
| Discussion|| |
Malodor and clinical signs of bleeding after removing the cover screws or abutments may be caused by infiltration of anaerobic bacteria through the implant -abutment interface. This phenomenon of infiltration can be assessed in two ways: Verifying the passage of bacteria to the inner part of a dental implant, ,,,, or in an inverted direction. ,,,,,
Bacterial infiltration from the outer to the inner part of the implant represents the best in vivo situation; however, when normal infiltration is evaluated in vitro, it can present certain disadvantages and problems. Normal infiltration is assessed by connecting the abutments to the implants under sterile conditions, immersing them in a bacterial suspension, and then looking for bacteria at the inner part of the implant.
The necessity of removing the prosthetic abutment from the implant limits the evaluation time of the current study; therefore, the samples were only evaluated after a certain period.
Most implant prosthetic components are composed of two parts: The abutment and the abutment screw; however, there are also one piece solid abutments that are screwed directly into the implants, and require no screws for retention. In the present research, as well as in other in vitro studies, ,,, the interface between the implant and the abutments that are in two parts were held partially immersed in a sterile solution because they exhibit two places of bacterial penetration (the interface between the abutment and implant and between the abutment and abutment screw hole). Quirynem et al.  found low interface contamination in 30 partly submerged implant-abutment assemblies when compared to those fully submerged. Other authors  sealed the upper part of the assemblies with a layer of gutta-percha and cyanoacrylate adhesive; however, the efficiency of these procedures was not found.
The methodology used in this current research was determined after several pilot studies. One of these pilot studies showed that assessment of the passage of bacteria to the internal part of implants may provide unreliable results, because external disinfection of the assemblies before the removal of the abutment from the implant is required to verify that bacterial infiltration occurred. Due to the difficulty in sealing that interface (implant-abutment interface), the disinfecting agent could infiltrate to the inner part of the implant by the same route of bacterial penetration, masking the results. Observations on bacterial infiltration into the implant  reported that no sample showed any infiltration with those results justified by the use of a chlorhexidine varnish. However, disinfection was carried out prior to the removal of the abutment for analysis, which could have masked bacteria viability. The same was observed in a past study by Dibart et al.,  in which the outer surface of the implant was disinfected to observe the inner part, and no infiltration was detected in any sample.
The verification of passage of bacteria in an inverted direction seems to be more reliable; however, external contamination may occur on account of extravasations of microorganisms after inoculation and placement of the abutment. ,, Jansen et al.  inoculated the inner part of 13 dental implant systems with various types of prosthetic connections using an E. coli suspension and found a large amount of conical connection samples that had external contamination; therefore, more than 50% of the samples were discarded.
Other studies  have demonstrated that there was no external contamination in 30 internal hexagon connection samples inoculated with 2 μL of Staphylococcus aureus suspension; all of the samples presented infiltration. Unlike the method used for verification of external contamination in the current study and other studies, , the authors of the previous study,  used paper cones to cover the outer portion of the implant-abutment interface, which may not have been an efficient method to assess the extravasation of the suspension, causing contamination of the external environment. Several studies , showed that, even though the same implant system is used (internal hexagonal implant-Ha-Ti, Mathys Dental Implants, Switzerland), the application of a chlorhexidine varnish on the implant-abutment interface did not originate infiltration in any sample, concluding that the varnish may have prevented bacterial penetration.
As bacterial penetration can occur in two directions, the present study evaluated penetration from the inverted direction by inoculating colonies of bacteria inside the implant. Thus, the number of samples with immediate external contamination was reduced, with one sample in G1 (external hex), two in G2 (hex indexed) and none in G3 (Morse taper) that presented external contamination.
Furthermore, to validate the current results, a bacterial viability test was performed in all samples that showed no infiltration after seven days. Those without bacterial viability were excluded, resulting in approximately 20% of the samples discarded in each group. The assessment of bacterial viability is an important stage, since it is not prudent to say that there was no infiltration without that analysis. Most studies ,,, showed no bacterial infiltration inside the implant and none of those studies identified any method to assess the bacterial viability of the inoculated implants.
A large variety of microorganisms seem to be able to infiltrate at the implant-abutment interface, with most of them, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivallis, Campylobacter rectus, Bacteroides spp., Fusobacterium spp. and Peptostreptococcus have been associated to peri-implantitis. , These microorganisms are small when compared to the openings found at the implant-abutment interfaces.
Escherichia coli is a Gram negative motile organism that is a rod-shaped bacteria with a 1.1 to 1.5 μm diameter and 2 to 6 μm length that provides ease of manipulation and is widely used in vitro studies to evaluate sterilization, disinfection and contamination, , its growth occurs in a short time (20 min) and it can be found in healthy oral cavities. For the reasons mentioned above, E. coli was selected to be used in this present study.
Jansen et al.  determined that 13 types of prosthetic connections presented bacterial infiltration. From that study, conical connection implant systems and the percentage of samples that had infiltration were: Ankylos system (Dentsply Friadent, USA) with 50%, the Astra system (Astra Tech, Sweden) with 69% and the Bonefit ITI implant system (Straumann, Switzerland) with 96%. These results are quite divergent from those obtained in the current study, in which three fixtures with Morse taper connections showed bacterial infiltration within the total sample size (n=40), representing an infiltration rate of 7.5%. Comparing the results of the external hexagon connections to those obtained in the present study, it was observed that 82% of the samples of the Branemark System (Nobel Biocare, Switzerland) exhibited bacterial infiltration, compared with 10.53% samples of the Conexão System (Conexão Sistemas de Prótese Ltda., Brazil). Other internal connections of different configurations also showed high bacterial infiltration rates, ranging from 38 to 100%, which is different from the current data, where the internal hex connection presented 4.88% samples with infiltration. The largest percentage observed in that study  is not related to a longer assessment period (14 days) as the current study presents, since bacterial infiltration occurred on the seventh day of follow-up. The unfavorable results obtained in that study can be related to the absence of torque used to assure proper abutment fit. The application of appropriate torque to abutment screws is important in improving the stability of the implant- abutment connection, while also influencing the size of the micro-gap at the implant-abutment interface. ,
Dye penetration through the implant-abutment interface of five implant systems was assessed at varying torque loads, 10 Ncm, 20 Ncm and the loads recommended by the manufacturer.  The results from that study indicated that microleakage decrease significantly when the applied torque was increased, which suggested that bacterial byproducts and nutrients required for bacterial growth are capable of passing through the interface gap, contributing to clinical malodor and inflammation of peri-implant tissues. 
Biological factors that contribute to successful oral rehabilitation with osseointegrated implants are also closely related to the mechanical characteristics of the prosthetic components. Three different types of prosthetic connections from the same manufacturer were evaluated in the current study and all exhibited similar bacterial infiltration behavior. Many manufacturers produce compatible prosthetic components of the same configuration, predominantly with the traditional external hexagon connection. In this present study, a small amount of samples showed infiltration along the implant-abutment interface. Torque was applied as indicated by the manufacturer, while electronic images that were captured through an optical microscope showed adequate adaptation of the connections. However, the test was performed without the application of masticatory loads that simulate a clinical situation. This fact may explain the low incidence of bacterial infiltration observed in the present research, in comparison to the results observed in other in vivo studies, ,,, where the instability of prosthetic connections under masticatory loads may have influenced the adaptation between all the components, causing gaps that allowed bacterial penetration.
In contrast to the current results, in which the percentage of infiltration for the external hexagon, internal hexagon and Morse taper connections was 10.53%, 4.88% and 7.5%, respectively; Steinebrunn et al.  proposed a new in vitro model to study microbial microleakage at the implant-abutment interface under dynamic forces using a chewing simulator. They obtained 100% bacterial infiltration in five implant systems evaluated.  The best performing connections were considered the most stable because they minimized micro-movements when loading forces were applied. Torque may have some influence on these results, considering that the external hexagon system required a torque of 45 Ncm, and a high number of dynamic cycles were necessary to promote bacterial infiltration, while the torque applied to the internal hexagonal system ranged from 20 to 35 Ncm. Moreover, the combination between the Morse taper connection and the internal hexagon showed the worst results, with the lowest average cycles, which may indicate that this combination of connections was not as efficient in promoting the stability of the assemblies. Therefore, it is presumed that bacterial infiltration at the implant-abutment interface occurs as a result of many factors, such as precision of fit and the degree of micro-movement between the implant and abutment, and torque forces used for connecting them. 
Dibart et al.  observed a micro-gap of less than 0.5 μm at the interface between the abutment and the implant with a Morse taper connection, which was considered insufficient to permit bacterial invasion. While using scanning electron microscopy (SEM) to analyze bacterial infiltration from the outer to the inner part, no bacteria were detected in the inner part after 24, 48 and 72 h. Those authors related their favorable results to the quality of the adjustment provided by Morse taper design.  While similar results were found in this current study, it is believed that the evaluation period proposed was insufficient, based on the fact that the turbidity of the medium in some samples was only observed after 72 h.
This current study evaluated the performance of different types of prosthetic connections to assess potential bacterial infiltration. The use of components from the same manufacturer allowed standardization of the material. The quality of the implant-abutment interface, as well as the stability of the prosthetic connection of various implant systems, may be factors directly related to the success of implants.
| Conclusions|| |
The following conclusions were drawn:
- A low level of bacterial contamination through the implant-abutment interface was evidenced in all study groups.
- Bacterial infiltration was similar in all types of prosthetic connections evaluated, despite the configuration of the interface between the implant and the abutment.
| Acknowledgments|| |
The authors thank Doctors Luciane Dias Oliveira and Antonio Olavo C. Jorge for their support of this study. The material used in this study were kindly supplied by "Conexão Sistemas de Prótese".
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[Table 1], [Table 2]