World Journal of Dentistry
Volume 15 | Issue 1 | Year 2024

Evaluation of Microbial Leakage in Three Different Implant Abutment Connections by Analyzing the Presence of Staphylococci: An In Vitro Study

Pranav Desai1, Paresh Gandhi2, Rajashree Jadhav3, Aarti Gachake4, Rupali Patil5, Pankaj Kadam6

1–6Department of Prosthodontics, Bharati Vidyapeeth Dental College and Hospital, Bharati Vidyapeeth (Deemed to be University), Pune, Maharashtra, India

Corresponding Author: Paresh Gandhi, Department of Prosthodontics, Bharati Vidyapeeth Dental College and Hospital, Bharati Vidyapeeth (Deemed to be University), Pune, Maharashtra, India, Phone: +91 9822309261, e-mail: pareshgandhi007@gmail.com

Received: 03 December 2023; Accepted: 06 January 2024; Published on: 20 February 2024


Aim: To evaluate and compare microbial leakage occurring in three different popularly used implant-abutment connections (IAC).

Materials and methods: Nine implant analogs and abutments have a Conelog connection (BioHorizons, United States of America), nine implant analog and abutments have a Conexa connection (B&B, Italy), and nine implant analog and abutments have an Internal octagon connection (Osstem, Korea) were immersed in bacterial peptone broth containing Staphylococci bacteria for 14 days. After this time, the implant analog abutment assemblies were disassembled, and swabs were taken from the internal surface of the connection, which were then cultured and evaluated for growth.

Results: Microbial leakage was seen in 15 out of 27 samples.

Conclusion: Higher quantities of bacterial ingress were seen in the Conexa and Internal octagon connections. However, the differences were not found to be statistically significant.

Clinical significance: The implant abutment connections is one of the most crucial components of the implant-prosthesis system, especially when it comes to the longevity of the treatment. When this IAC is subjected to occlusal loading, micromovements of the abutment occur, which perpetuate a microgap between the abutment and implant. This microgap can cause leakage of microorganisms, which would then act as a bacterial reservoir. This gap is located at the level of the alveolar crest. This could lead to unfavorable biological consequences such as inflammation and infection, leading to peri-implantitis and increased crestal bone loss, which could ultimately lead to implant failure.

How to cite this article: Desai P, Gandhi P, Jadhav R, et al. Evaluation of Microbial Leakage in Three Different Implant Abutment Connections by Analyzing the Presence of Staphylococci: An In Vitro Study. World J Dent 2024;15(1):6–12.

Source of support: Nil

Conflict of interest: None

Keywords: Dental implants, Implant-abutment connection, Implant-abutment microgap, Peri-implantitis


The long-term success of implant restoration is frequently achievable with proper surgical and prosthetic protocols. A cumulative long-term success rate of 94.6% has been reported by Wang et al.1 An estimated 4.42% of implants failed during function due to peri-implantitis or other biological or mechanical reasons. Often, the etiology of peri-implantitis is multifactorial. The presence and size of the microgap present a major risk factor contributing to microbial leakage and successful implant survival.

Rapid progress in research and development of dental implant technologies has given rise to a variety of innovative implant designs, predictable surgical protocols, and various treatment options, which help us choose a suitable approach to treat a particular case. Two-stage implants consist of an abutment which connects to the underlying fixture by means of a geometric (frictional) connection and an abutment screw. When this implant-abutment system is subjected to occlusal loading, the adaptation of the abutment to the implant connection may be affected, leading to the development of microscopic gaps in the implant-abutment connection (IAC).2 This microgap can cause leakage of microorganisms, which would then act as a bacteriological reservoir. This gap is located at the level of the alveolar crest. This could lead to unfavorable biological consequences such as inflammation and infection, leading to peri-implantitis and increased crestal bone loss, which could ultimately lead to implant failure.3 It has been proven in previous studies that the Morse Taper connection shows fewer amounts of microbial leakage through the implant-abutment connection IAC.4 The present study, therefore, compares three connections having different degrees of tapers in an attempt to identify a suitable connection and taper. Such a study comparing these specific connections has not been attempted before. Thus, the aim of the current study was to compare and evaluate the amount of microbial leakage occurring between three popularly used implants to choose the correct one for use in clinical practice.

Prevention of microbial leakage at the IAC is important in order to minimize inflammatory reactions and maximize crestal bone stability.


An in vitro study design was conducted on three different types of implant-abutment assemblies to evaluate the amount of microbial leakage.

Total sample size was estimated using following formula:

n = [(Z1 – α)s]2 / L2

Level of significance (α) = 95% Z1 – α = 1.96

Power of study (β) = 80% = – 0.84

S (standard deviation) = 9.5

Precision (L) = 6.3

The values for calculating this were substituted from a study by Khorshidi et al.5 Based on the calculation, the sample size of 18 was calculated. An extra nine samples were tested so that the sample size requirement was met at the end of the study to compensate for any experimental errors during the study. The IRC approval number for the present study was BVDU/DCH/734/2020/21. The study was done in July 2022 and lasted for 24 days, from the incubation of the samples to the microscopic colony count for analysis of growth. The flow diagram of the study method is illustrated briefly in Flowchart 1.

Flowchart 1: Flow diagram of study method

The study was divided into the following steps:

Preparation of samples.

Preparation of culture test specimen.

Evaluation, statistical analysis, and verification of bacterial leakage.

Preparation of Samples

The implants to be studied were divided according to their connection system. They were divided into three groups, each group containing nine implant analog abutment assemblies of the same geometry.

Group I: Nine implant analogs–abutment assemblies with an Internal octagon connection (Osstem).

Group II: Nine implant analog–abutment assemblies with a Conexa connection (B and B).

Group III: Nine implant analog–abutment assemblies with a Conelog (BioHorizons).

The implant analogs were retrieved from their sterile packaging with the help of sterile pliers. The abutments were carefully connected to the implants and tightened using the respective torque ratchet according to the manufacturer’s instructions (Fig. 1). These implant analog–abutment assemblies were used as samples for testing the microbial leakage through their IAC. Nine such samples were made for each of the three groups.

Fig. 1: Abutment, implant analog, respective hex drivers, and torque wrenches for the three groups

Preparation of Culture Test Specimen

Test specimens of Staphylococcus aureus were prepared on blood agar plates, which were confirmed by a positive coagulase test. An inoculating loop was heated on a Bunsen burner, allowed to air cool, and then used to pick up bacterial colonies from the blood agar plate. This inoculating loop with bacterial colonies was inserted in sterile test tubes filled with 4 mL of peptone water at room temperature and compared to a 0.5 McFarland standard to adjust the turbidity of the bacterial suspension. The assembled implant analog–abutment samples were then completely immersed in 4 ml of bacterial suspension and placed in an incubator at 37°C for 14 days.

Evaluation and Verification of Bacterial Leakage

After 14 days of incubation, the samples were removed from the test tube using sterile pliers, wiped with 70% alcohol using a cotton ball, and dried with sterile gauze. This procedure ensured the sterility of the outer surface of the samples without affecting bacterial viability on the inside. Next, the specimens were carefully disassembled using respective hex drivers, and the inner surface of the implant analogs was sampled for bacterial contamination using sterile paper points (Fig. 2). These paper points were streaked on blood agar plates and incubated at 37°C for 48 hours.

Figs 2A and B: (A) Nine implant analog assemblies; and (B) Corresponding paper point swabs obtained

After incubation of the samples, the growth of colonies in the culture medium was evaluated. From the blood agar plates, which showed the growth of organisms (Fig. 3), colonies of bacteria were picked up using inoculating loops and then placed on sterile glass slides for gram staining. The slides were then observed under a compound microscope (Fig. 4). The microscopic appearance of Staphylococcus species is cocci in grape-like clusters. After microscopic examination, a coagulase test using plasma was carried out to verify and confirm the presence of Staphylococcus aureus in the inner part of the assembly.

Fig. 3: Growth of colonies from a prepared sample

Fig. 4: Gram-staining revealed the presence of gram-positive Staphylococci

The statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) software, version 21, SPSS Inc (Chicago, Illinois, United States of America) to analyze the results.


Nine samples of each of the Conexa, Conelog, and Internal octagonal connection implant analogs with the abutments were evaluated for the presence of microbial leakage. The occurrence of microbial leakage was individually calculated for each sample after a time period of 14 days. The presence of leakage into the implant abutment interface indicated the presence of microgaps.

In the present study, 15 out of 27 samples indicated that microbial leakage had taken place. The results were tabulated and analyzed. Table 1 shows the result of the normality test using the Shapiro–Wilk test. Since the p-value of two groups out of three was found to be <0.05, the data were not normally distributed. Nonparametric tests were used for comparative statistics between the three groups. Table 2 provides the descriptive statistics related to the three implant-abutment interfaces. The mean value of the Internal octagon connection (Osstem) was found to be 6.556, with a standard deviation of 9.20. Figure 5 shows the distribution among samples for the Internal octagon group. Similarly, for the Conexa connection (B and B), the mean value of the colony-forming unit was 11.444, the standard deviation was 13.24, and it was the highest among the three groups. Figure 6 shows the distribution among samples for the Conexa group. Lastly, for Conelog (BioHorizons), the mean ± standard deviation was found to be 5.222 ± 8.68. Figure 7 shows the distribution among samples for the Conelog group. Table 3 shows the comparison between the three groups (group I vs group II vs group III) conducted using the Kruskal–Wallis test, as the data was not normally distributed. Figure 8 shows the comparison of mean and standard deviation among the three groups. The p-value was found to be greater than 0.05, which was indicative of the fact that the mean difference between the three groups was not statistically significant. Therefore, no statistical inferences can be drawn on the basis of the colony-forming units between the three groups. Table 4 shows the results of the Mann–Whitney U test, which was done to compare the two groups individually. The Conelog (BioHorizons, United States of America) performed marginally better in terms of less incidence of microbial leakage compared to the other two groups. However, this comparison of mean differences between group I and group II was not found to be statistically significant. Similarly, the other two comparisons (group I vs group III and group II vs group III) were found to be statistically insignificant as well.

Table 1: Microbial leakage in groups I, II, and III after 14 days
Sample Group I (no. of colonies) Group II (no. of colonies) Group III (no. of colonies)
Sample 1 0 18 2
Sample 2 0 1 3
Sample 3 0 0 0
Sample 4 0 8 0
Sample 5 0 0 0
Sample 6 14 0 0
Sample 7 23 13 2
Sample 8 4 36 16
Sample 9 18 27 24
Table 2: Normality testing using Shapiro–Wilk test
Tests of normality
Kolmogorov–Smirnova Shapiro–Wilk
Statistic Degree of freedom (df) Significance Statistic df Significance
Group I (Osstem) 0.317 9 0.009 0.748 9 0.005
Group II (Conexa) 0.229 9 0.190 0.855 9 0.085
Group III (Conelog) 0.379 9 0.001 0.669 9 0.001

aLilliefors significance correction

Table 3: Descriptive statistics of microbial leakage using the three implant-abutment interface using colony forming unit as a parameter
Minimum Maximum Mean Standard deviation
Group I: Internal octagon connection (Osstem) 0.00 23.00 6.556 9.20
Group II: Conexa connection (B and B) 0.00 36.00 11.444 13.24
Group III:
Conelog (BioHorizons)
0.00 24.00 5.222 8.68
Table 4: Comparison of microbial leakage between the three groups using the Kruskal–Wallis test
Mean rank Kruskal–Wallis H-value p-value
Group I: Internal octagon connection (Osstem) 12.89 1.048 0.592
Group II: Conexa connection (B and B) 16.11
Group III: Conelog (BioHorizons) 13.00

Fig. 5: Distribution of samples along with colony-forming units for group I (Osstem)

Fig. 6: Distribution of samples along with colony-forming units for group II (Conexa connection)

Fig. 7: Distribution of samples along with colony-forming units for group III (Conelog, BioHorizons)

Fig. 8: Comparison of mean and standard deviation of the three groups of implant-abutment interface

The observation for all the samples is tabulated in Table 5.

Table 5: Post hoc analysis for comparison between two individual groups using Mann–Whitney U test
Mann–Whitney U value Z-value p-value
Group I: Internal octagon connection (Osstem)
Group II: Conexa connection (B and B)
31.000 0.878 0.436
Group I: Internal octagon connection (Osstem)
Group III: Conelog (BioHorizons)
40.000 0.047 0.962
Group II: Conexa connection (B and B)
Group III: Conelog (BioHorizons)
31.000 0.865 0.387


The present study was designed to compare the amount of microbial leakage that occurred through the implant-abutment interface in three different implant systems. The occurrence of bacterial leakage through the IAC has been shown in several in vitro studies in both static and dynamic loading conditions.6 According to a study done by Quirynen et al.,7 microbial leakage was seen in the IAC when the implants and abutments were kept in a medium inoculated with oral microorganisms. Jansen et al.8 reported microbial leakage using E. coli. Bacteria in 13 different IAC. Nassar et al.9 conducted a comparison between internal hexagons and trilobed implants in a manner similar to this study. Another DNA probe analysis by Callan et al.10 showed the presence of pathogenic periodontal bacteria in the implant abutment microgap. Assessment of the risk of invasion of bacteria in internal Morse taper and trichannel internal connections was done by Tesmer et al.,11 and they found that Morse taper connections demonstrated a lesser amount of microbial penetration through the IAC. Since it has been established that tapered connections perform better to resist microbial leakage compared to parallel walled internal connections, one aim of this study was to further compare different tapers to try and identify the degree of taper most suited to resist microbial leakage. The time period of incubation was 14 days, according to the results published by Koka et al.,12 who stated that it was the approximate time in which microbial ingress took place. Nakazato et al.13 demonstrated a much shorter time of just 4 hours for bacterial leakage to occur. Nassar et al.9 conducted a similar comparison between internal hexagons and trilobed implants.

This study was therefore conducted to identify a suitable implant system that demonstrated minimal microbial leakage. The IAC is one of the most crucial components of implant restoration to fulfill the treatment objectives of prosthesis success and longevity. Any micromovements taking place at the implant and abutment junction should be eliminated. Traditionally used external connection implants had a few major drawbacks, such as lack of height to resist nonaxial loads, frequent screw loosening, and component fracture.14

Among the two-piece implant systems, the internal hexagonal connection, in which a portion of the abutment is incorporated into the implant body, is currently the most popular. The fact that this connection guarantees appropriate abutment seating, anti-rotational engagement, resistance to lateral stresses, and great esthetic results most certainly accounts for this. According to reports, compared to the external connection, this connection is less conducive to fluid penetration.15 Different internal connections have been found to be more stable under loading conditions than external connections, which, when subjected to functional loading, encourages the abutment to move slightly and, as a result, causes bacterial leakage

Eventually, the popularity of Morse taper and friction fit implants gave rise to multiple manufacturers introducing their proprietary connection designs with various claims of benefit.4 Thus, three popular connection geometries were chosen to be evaluated, namely, the internal hexagon, Internal octagon, and conical connections. Most connections in the market used today vary from 5 to 15° of internal taper. Thus, it was chosen to evaluate internal tapers of 5, 7.5, and 8° to represent various connections that feature similar tapers.

Conelog® screw-line implants (Fig. 9A) are equipped with a 7.5° internal taper and a conical connection for reliable transfer of force and torque and the three proven CAMLOG grooves for precision abutment positioning. Clearly, perceptible tactile feedback lets the user know when the abutment is positioned correctly by the three grooves and apical external taper. The Conelog connection allows for greater precision in the placement of the abutment, ensuring a more accurate and esthetically pleasing result. Com-octa implants and abutments (Fig. 9B) feature a connection that is an Internal octagon and 8° internal taper. The octagonal design creates a more secure connection between the implant and the abutment, providing improved stability and reducing the risk of loosening over time. The Conexa connection (Fig. 9C) features a 5° internal taper and an internal hexagon. This connection also features a cold weld that occurs instantaneously between the abutment and the implant when the abutment is torqued onto the implant. This reduces the possibility of screw loosening and reduces the amount of microleakage. A specialized extractor tool is used if the abutment has to be removed from the implant.

Figs 9A to C: Schematic cross-section of the BioHorizons, Osstem, and B and B implants, respectively

Oral bacteria include Streptococci, Lactobacilli, Staphylococci, Corynebacteria, and various anaerobes, in particular Bacteroides. Oral microflora is most commonly found in gingival crevices, coronal plaques, the dorsum of the tongue, buccal mucosa, and saliva. In a study by Ohara-Nemoto,16 the occurrence of staphylococci in the oral cavity was examined in healthy adults. The results showed that Staphylococcus aureus was the most frequently found species in the oral cavity, followed by Staphylococcus epidermidis and others. Since Staphylococcus aureus is a common resident of the oral microflora, it was chosen in the study to check for bacterial leakage. Similar results were found in this study, consistent with the present literature.

The behavior of the microgap in response to mechanical stress and cyclic loading must not be ignored. Micromovements often occur, increasing the magnitude of microbial leakage seen in the IAC. Further studies are required in this field to account for the dynamic loading of the implant restoration system in the oral environment. Future studies may also consider the use of different complexes of organisms found in the oral cavity rather than individual organisms or performing an in vivo assessment of microbial leakage after subjecting the IAC to cyclic physiologic loading. Such a study may also be done to evaluate differences in microbial leakage in patients with parafunctional habits such as clenching and bruxism to evaluate the effect of occlusal overload on microbial leakage at the level of the IAC. Clinicians must carefully evaluate and assess the options available and identify a proper system that shows the least amount of microbial leakage and preferably has an internal taper of the vertical connection walls. Furthermore, cold-welded IAC may be more favorable in reducing mechanical complications such as screw loosening.


Within the limitations of an in vitro study, it was seen that microbial leakage occurred in all three types of IAC. The Conelog connection implant showed the least amount of microbial leakage, followed by the Conexa and the Internal octagonal connection implants. However, the comparison between the amount of microbial leakage that was seen in the Internal octagonal, Conexa, and Conelog implant connection systems was statistically insignificant.


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