|Year : 2020 | Volume
| Issue : 1 | Page : 22-34
An in vitro evaluation of microleakage in platform-switched implants at implant–abutment interface by contamination assessment of implant wells and respective abutment surfaces
V Yamuna1, Roseline Meshramkar1, RD Kulkarni2, Manjunath A Hosamani2, K Lekha1, Ramesh K Nadiger1, Nagarajan Chidambaram1
1 Department of Prosthodontics, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka, India
2 Department of Microbiology, SDM Medical College and Hospital, Dharwad, Karnataka, India
|Date of Submission||03-May-2019|
|Date of Acceptance||13-Nov-2019|
|Date of Web Publication||08-Jul-2020|
Dr. V Yamuna
Department of Prosthodontics, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background and Objectives: In contemporary implant dentistry, the success of implant treatment is assessed by measuring the crestal bone level apart from osseointegration. Peri-implant bone plays a vital role in the esthetics of implant restorations. With loss of peri-implant bone, soft tissue loss happens which eventually compromises the esthetics and mechanical properties of restorations. To prevent marginal bone loss, many inventions are made by modifying the implant designs, implant–abutment connections, and techniques. Platform-switched concept is one such invention evolved to prevent peri-implant bone loss. This beneficial effect of platform-switched implants was studied by many researchers. However, few studies were reported in the literature on microleakage in platform-switched implants. Thus, the purpose of this study is to evaluate microleakage at implant–abutment interface in platform-switched implants.
Materials and Methods: Fifteen in-built platform-switched implants and corresponding abutments with internal hexagonal design were connected using screws. After the confirmation of the sterility of the implants and abutments, the assemblies were incubated in brain–heart infusion broth inoculated with Staphylococcus aureus for 14 days at 37°C. After 14 days, the implants and abutments were disassembled. Samples were collected from three different sites, i.e., walls of the wells of the implants, the deepest portion of the wells of the implants, and the surface of the abutments with help of paper points. Using the samples, colony counting and Gram staining were done to evaluate the microleakage at the implant–abutment interface.
Results: Microbial contamination was found to be present at all the sites from which samples were collected.P < 0.05 was found when the different sites were compared with each other. The abutment surface found to have the least contamination, and the walls of the implant wells found to have the highest contamination.
Conclusion: Within the limitations of this study, it was concluded that microleakage is present in the platform-switched implants with screw-retained internal hexagonal connections at the implant–abutment interface.
Keywords: Implant–abutment interface, internal hexagonal connection, platform-switched implants, screw retained
|How to cite this article:|
Yamuna V, Meshramkar R, Kulkarni R D, Hosamani MA, Lekha K, Nadiger RK, Chidambaram N. An in vitro evaluation of microleakage in platform-switched implants at implant–abutment interface by contamination assessment of implant wells and respective abutment surfaces. J Dent Implant 2020;10:22-34
|How to cite this URL:|
Yamuna V, Meshramkar R, Kulkarni R D, Hosamani MA, Lekha K, Nadiger RK, Chidambaram N. An in vitro evaluation of microleakage in platform-switched implants at implant–abutment interface by contamination assessment of implant wells and respective abutment surfaces. J Dent Implant [serial online] 2020 [cited 2020 Sep 19];10:22-34. Available from: http://www.jdionline.org/text.asp?2020/10/1/22/289230
| Introduction|| |
The prime goal to reduce peri-implant crestal bone loss led to the discovery of the concept of platform switching into implant dentistry. Platform switching refers to the use of smaller-diameter abutment on larger-diameter implant collar. Due to inward repositioning of the implant–abutment interface in platform-switched implants, the peri-implant bone loss is less. Added to that, the stress concentration area is away from the cervical bone–implant interface.
In 2006, Lazzara and Porter introduced this amazing platform-switching concept into implantology for the very first time. They described the concept as an inward metal ring in the coronal part of the implant that is in continuity with the alveolar bone crest.
The various advantages of platform-switched implants are shifting the stress concentration away from the bone–abutment interface, crestal bone preservation, and controlled biological space reposition which leads to improved esthetics of the restoration.
The platform-switched implant system is of great significance because peri-implant bone level is the paramount factor in determining implant success. As stated above, one of the advantages of platform-switched implants is the preservation of peri-implant bone. The platform-switched concept has two-stage implant system like other nonplatform-switched systems. The implant–abutment interface of nonplatform-switched system is prone to microleakage and bacterial trap. This will in turn cause inflammatory reaction in peri-implant soft tissue and interfere with osseointegration by causing peri-implant bone loss in nonplatform-switched system. One of the prime causative factors for peri-implantitis is microleakage along the implant–abutment interface.
Microleakage is defined as leakage of minute amount of fluids, debris, and microorganisms through the microscopic space between a dental restoration or its cement and the adjacent surface of the cavity preparation.
A number of investigators tried to quantify bacterial leakage of different implant systems using microorganisms such as Staphylococcus aureus or molecules such as endotoxin, rhodamine B, toluidine blue, and gas flow. In spite of various techniques, microbiological tests are always gold standard. Bacterial microleakage tests conducted in the earlier studies used various bacteria, i.e., from facultative to obligate anaerobes, and their size ranges from 1 to 10 μm. The studies also analyzed leakage from the inner parts of the implants to the outside parts, from external portion to the internal parts of an implant or using qualitative and/or quantitative methods, including turbidity analysis of nutritional broth and bacterial DNA analysis. The problems of false-positive or false-negative results are not uncommon due to reasons such as use of forceps or pliers to fix the implants, freehand inoculation of bacterial broth into the implants, total coating of the implants, use of same torque wrench for several samples, lack of knowledge about the implant's internal volume, the bacterial type as well as the disinfection procedure followed to evaluate fluid flow orientation. Studies used various implant systems such as Nobel Biocare, Straumann, Bicon, Ankylos, Astra Tech, Neodent, and Branemark. Regardless of all the implant systems, the degree of bacterial penetration in specific implant system depends on precision of fit between the implant and the abutment, degree of micromovement between the components, and torque forces to connect them.
Contemporary treatment in dentistry involves implant restorations majorly. This is because of the success that implant enjoys from osseointegration. Still, failures in implant therapy are recorded on account of mechanical and biological consequences. Mechanical nonperformance happens due to loosening or fracturing of screw, rotation of abutment, and loss of developed preload in the screw between the abutment and the fixture. Biological stumbling blocks such as mucositis and peri-implant bone loss have been seen. Remarkable bone loss has been reported with two-piece implants by various studies. In case of two-piece implant systems, gap between implant and abutment are inevitable. This microgap is the principal reason for peri-implantitis by inflammatory reactions.
As mentioned previously, the platform-switched implants are two-piece systems. In this platform-switched implant, very few researches have been conducted to evaluate microleakage. Therefore, the aim of the present study is to investigate the microleakage in platform-switched implants at implant–abutment interface.
| Materials and Methods|| |
This study was conducted to evaluate the microleakage in platform-switched implant system at the implant–abutment interface in the:
- Department of Prosthodontics, SDM College of Dental Sciences and Hospital, Dharwad
- Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad.
Armamentarium used for the study
- Dental implant (Touareg™-OS, Adin Dental Implant System Limited, Israel) [Figure 1]
- Titanium abutment (Adin Dental Implant System Limited, Israel)
- Biosafety cabinet 2A2 (Alpha Linear, Bangalore) [Figure 2].
- Sterile brain–heart infusion (BHI) broth
- Test tubes
- Broth tubes with S. aureus
- Paper points
- Finger key [Figure 3]
- MIS - calibrated manual torque wrench [Figure 3]
- BHI agar plates
- Gram stains.
Implants and abutments
The study was conducted using 15 Touareg™-OS implants of dimension 4.2 mm diameter and 11.5 mm length from Adin Dental Implant System Limited. Touareg™-OS implants are tapered spiral implants that condense the bone during placement and provide immediate stability. The prosthetic connections of this implant system are a standard internal hex 3.5 mm diameter. The advantages of these implants are double lead threads, high primary stability, self–drilling, and self-cutting with built-in platform switching. The abutments were also purchased from Adin Dental Implant System that corresponds with the Touaraeg™-OS implants. The abutments are made of Ti Grade 5 and belong to RS Slim Titanium Abutment 3 mm.
This study involved platform-switched implants and abutments that were connected and incubated in S. aureus broth to check the microleakage at the implant–abutment interface.
Implant and abutment sterility test
Fifteen platform-switched implants with internal hexagon design and abutments were taken. The abutments were connected [Figure 4] with the implants using finger key and torqued to 30 Ncm [Figure 5] inside a biosafety cabinet 2A2. Each abutment along with the connected implant was dropped in individual test tubes containing sterile BHI broth [Figure 6] and [Figure 7]. They were incubated for 72 h. After 72 h, the tubes were checked for any turbidity. A sterile BHI broth test tube was used as control tube. Absence of turbidity in all the experimental tubes revealed that all the implants and abutments were sterile and fit to use for the further steps in this research. The implants and abutments were cleaned with autoclaved distilled water thoroughly to remove the BHI broth from them for further use [Figure 8] and [Figure 9].
|Figure 6: 15 assembled implants and abutments dropped in sterile brain–heart infusion broth|
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|Figure 8: The assembly removed from the sterile brain–heart infusion broth|
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Preparing the implant and abutment assemblies for incubation inStaphylococcus aureus broth
The implant and abutment assemblies were dropped in 15 test tubes of BHI broth inoculated with S. aureus [Figure 10],[Figure 11],[Figure 12]. The assemblies were kept inside the incubator for 14 days at 37°C [Figure 13],[Figure 14],[Figure 15]. One separate tube with BHI broth inoculated with S. aureus was kept as control.
|Figure 10: The assembly was dropped in tube with brain–heart infusion broth|
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|Figure 12: Inoculation of Staphylococcus aureus in the sterile brain–heart infusion broth with the assembly|
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Collection of samples
After 14 days, the assemblies were taken out and immersed in 70% alcohol for 3 min to sterilize the surface [Figure 16]. Then, the assemblies were dried completely. The implants and the abutments were disassembled under sterile conditions in a biosafety cabinet 2A2 [Figure 17]. Samples were collected from three different sites of an assembly using paper points: Site A – The sample from the walls of the abutments [Figure 18]; Site B – The sample from the walls of the wells of the implants [Figure 19]; Site C – The sample from the deepest part of the wells of the implants [Figure 20].
|Figure 19: Sample collection from the walls of the wells of the implants|
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|Figure 20: Sample collection from the deepest portion of the well of the implant under × 3.5 magnification|
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Contamination assessment from the collected samples
The paper points were placed in sterile BHI broth and vortexed. 100 μL of the vortexed broth was pipetted out immediately and evenly spread on BHI agar all over the plate [Figure 21] and [Figure 22]. The plates were incubated at 37°C for 72 h [Figure 23]. After 72 h [Figure 24], colonies were counted [Figure 25] and cross-verified using Gram stain. Meanwhile, the tubes were incubated further for 72 h to assess contamination. The tubes showing growth were subjected to Gram stain to confirm the growth of S. aureus. Thereby, cross-contamination was ruled out during the procedure.
|Figure 21: Sample dropped in sterile brain–heart infusion broth and vortexed|
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|Figure 23: Incubation of brain–heart infusion broth with samples for 72 h|
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Evaluation of the colonies
The BHI agar plates were incubated at 37°C for 72 h. The S. aureus was identified using the following nature of S. aureus presentation in BHI agar plates after 72 h of incubation, i.e., 7 mm or more in diameter, creamy opaque with variable yellow or golden color colonies [Figure 26].
Gram stain of Staphylococcus aureus
S. aureus can be characterized by round, purple Gram-positive bacteria that were presented in clusters as a bunch of grapes.
Data analysis was performed using SPSS software (version 20, IBM, Armonk, NY, USA). The values of colony counting units were tabulated [Table 1]. Those values were used for descriptive statistics, i.e., mean and standard deviation. Paired t-test was used to find the level of significance. Statistical significance was set to P < 0.05.
Ethical approval date and registration number: November/15,/2016, and 02_D018_70774.
| Results|| |
The present study was designed to evaluate the microleakage at the implant–abutment interface with platform-switched implants. The data obtained during the study, as shown in [Table 1], were subjected to statistical analysis using SPSS software (version 20, IBM, Armonk, NY, USA). An overview of the results is shown in [Table 2], [Table 3], [Table 4]. The mean value and standard deviation are tabulated in [Table 3]. The result was analyzed using “paired t-test” in which the significance level was set as P < 0.05, as shown in [Table 4].
The mean and standard deviation of Site A, Site B, and Site C are tabulated in [Table 3]. The mean and standard deviation of comparisons among the various sites are shown in [Table 3]. The contamination caused by microleakage found to be present in all the sites. Site B was more contaminated than the other two. This is understood by the higher mean and standard deviation values of Site B when compared with Site A and Site C. Among Site A and Site C, Site C was more contaminated than Site A and this can be understood by higher mean and standard deviation values of Site C than Site A. A P value of all the pairs, i.e., Site A and Site B, Site B and Site C, and Site C and Site A, found to be <0.05. Therefore, it is statistically significant and shows the presence of microleakage at the implant–abutment interface in platform-switched implants.
At the time of reporting, it was revealed that the implant nos. 1, 2, and 5 were completely sterile.
The plates showing no growth but the broth tube showing turbidity were seen in some tubes as indicated. Broths from these tubes were subjected to Gram stain to confirm the growth of S. aureus to rule out contamination during the procedure.
Of the total implants subjected to testing, 12 showed leakage while three did not show leakage.
| Discussion|| |
Titanium dental implants are uniquely used in the management of patients with edentulous arches, either partial or complete. The dental implants are available in plenty of materials with different platform designs, diameter, body shape, length, and surface coatings. Studies documented that long-term success of implant treatment depends on the marginal bone level changes. Few possibilities for the marginal bone loss are surgical trauma, occlusal overload, microgap, peri-implantitis, biologic width, and implant–abutment connection.
For the preservation of the marginal bone, various advancements have been made through researches. One of the advancements was the use of wider diameter implants and narrow diameter abutments, platform-switched concept. This concept was investigated by various investigators. Lazzara and Porter, 2006 commented on the advantage of lesser bone loss in platform-switched implants than the regular platform implants.
After the study of Lazzara and Porter on platform-switched implant concept, many researches were carried out to find the benefit of platform-switched implants on crestal bone loss.
Canullo et al. through their research have found that bone loss around platform-switched implants to be lesser because the platform-switched implants shift the stress concentration away from the peri-implant bone.
Yun et al. conducted a short-term clinical study on marginal bone-level changes around microthreaded and platform-switched implants. They have reported that marginal bone loss was less and also good short-term implant survival rate with microthreaded and platform-switched implants.
A literature search was done on implant platform -switching concept by Cumbo et al. They have found the capability of reducing or eliminating crestal bone loss by platform-switched implants.
Guerra et al. carried out a multicentered randomized clinical trial between platform-switched and nonplatform-switched implants in the posterior mandible. The findings of the study have found to be favorable for platform-switched implants on the maintenance and even enhancement of crestal bone level.
Chrcanovic et al. have stated that the marginal bone loss around platform-switched implants was less than nonplatform-switched implants through literature search.
Liu and Wang studied the beneficial effect of platform-switched implants on preservation of crestal bone. They have detected that microleakage is inevitable because microgap and micromovement at implant–abutment interface are unavoidable. The implant–abutment interface of platform-switched implants shifts the microleakage away from the crestal bone.
A retrospective study was conducted by Nayak et al. They have reported that by shifting the platform, the implant–abutment interface will be shifted away from the crestal bone. However, it does not affect the microgap. The fit between the implant and the abutment have found to be more important.
Microgap is found to be one of the most common risk factors for marginal bone loss. It should be taken care for preservation of marginal bone, which determines implant success. Microgap is inevitable when two components fitted together, i.e., implant and abutment. Broggini et al. and Enkling et al. through their research have found that microgap and microleakage are one of the etiologies for early marginal bone loss. This statement was further strengthened by Tsuge et al. They have observed that the microgap is influenced by the design of the implant–abutment interface. Also suggested that microgap can be a source for peri-implantitis through microbial contamination.
Efforts have been made by many studies to measure the microgap between the implant and the abutment. Tsuge et al. have reported that both horizontal and vertical discrepancies occur in the implant–abutment marginal fit. The microgap between the implant and the abutment ranges from 2.3 to 5.6 μm. Scarano et al. have stated that microgap in screw-retained implant–abutment system is critical for bacterial colonization and the microgap size will be much largerin vivo than seen in vitro.
The leakage tests have been used in dentistry since long time. There are various leakage tests such as rhodamine dye penetration test and endotoxin penetration test. However, bacterial leakage test was found to be gold standard and most commonly used method to check microleakage in dentistry. Bacterial leakage tests are based on the microgap size between the components and passive as well as active bacterial diffusion into the microgap of assembled test components.
Among the bacterial leakage studies, various pathogens include Aggregatibacter actinomycetemcomitans, Escherichia coli, and S. aureus. Studies use S. aureus because of their ease of culture as said in a study by Teixeira et al.
Many studies were carried out to reduce the microgap and thereby microbial contamination. Various implant–abutment connection designs such as connection with or without self-inhibition, with mandatory index and combination of cone and index in conjugation with various prosthetic platforms in implant dentistry, which comprise external hexagon, internal hexagon, Morse cone, and platform switching were evolved in an effort to decrease the microgap between the implant and the abutment. The platform switching is mainly indicated for single restoration with reduced prosthetic space for crestal bone and papilla preservation.
Berberi et al. studied leakage between the implant–abutment connection among three implant systems using rhodamine B dye penetration. One of the three implant systems were platform switched and they have reported the presence of leakage with platform-switched implant system at the implant–abutment connection.
Screw joint can be defined as the use of screws to connect the implants with the abutments. They are tightened using torque wrench. Application of torque can be achieved either manually or mechanically fabricated tools. Many studies were carried out to analyze the influence of the closing torque values on the microleakage at the implant–abutment connections.
Larrucea Verdugo et al. studied the effect of torque values on microleakage at implant–abutment connection. They have found decreased microleakage with increased torque. Application of manufacturer recommended torque found to reduce microleakage.
Gross et al. studied the effect of various closing torque values on microleakage with screw retained implant–abutment connections. They found decreased microleakage with increased recommended torque values. Therefore, they decrease the complications of microleakage.
The current study evaluated microleakage at the implant–abutment interface in platform-switched implants. All the implants that were used in this study had in-built platform switching and connected at 30 Ncm torque with abutments by screw retained internal hexagonal connection. The microleakage was assessed using S. aureus microorganisms by collecting samples from three different sites – Site A: Walls of the abutments, Site B: Walls of the wells of the implants, Site C: The deepest portion of the wells of the implants. Therefore, a total of 45 samples used, i.e., 15 samples from each site.
The results obtained from the present study indicated that microleakage was present in 12 samples. Three samples out of 15 tested were completely sterile without microleakage at implant–abutment interface in platform-switched implants. Statistically, there was significant contamination in all the sites from which samples were collected due to microleakage at implant–abutment interface in platform-switched implant system.
Connection design used in the present study to evaluate microleakage was internal hexagonal connection and found the presence of microleakage. This is in consistent with the findings of the study done by Nassar and Abdalla where they used similar study protocol. They have stated that microleakage between the implant and abutment is inescapable. The amount of microleakage depends on the design of the implant abutment connection. Added to that internal hexagon showed more microleakage than trilobed internal connection.
Teixeira et al. used S. aureus and internal hexagon connections similar to the present study and have reported the presence of microleakage with internal hexagonal connections.
D'Ercole et al. compared microleakage between internal hexagonal connections and conical connections. They have described more microleakage with internal hexagonal connections than conical connections even when narrow abutments connected to wider platform implants.
A study by Tripodi et al. where they compared internal hexagonal implant–abutment connection with other connections and have reported that internal hexagonal connections with more microleakage than the other comparative connection groups. Also found early occurrence of microleakage with internal hexagonal connections than Morse taper implant–abutment connections.
In 2016, Gherlone et al. conducted anin vitro study to evaluate resistance against bacterial leakage of new conical implant–abutment connection and conventional connections. They also have found the presence of microleakage with internal hexagonal connection.
da Silva-Neto et al. have found that the highest microleakage with internal hexagonal connections than external hexagonal connections and Morse-tapered connections.
Studies,,,, have found the presence of microleakage with internal hexagonal connections. As stated previously, microleakage may cause peri-implant bone loss and peri-implantitis. In spite of that, the use of the internal hexagonal connections with platform-switched implants was proved mechanically beneficial over external hexagonal connections by Freitas-Júnior et al. through a three-dimensional finite element analysis and anin vitro study.
The present study used screws to connect the implants with the abutments and found the presence of microleakage. This result is similar to the findings of research by Assenza et al. They conducted anin vitro evaluation of bacterial microleakage in implants with different implant–abutment connections included screwed trilobed connection, cemented connection, and internal conical connection. They have found the presence of more microleakage with screwed implant–abutment connections than the other two groups.
The presence of microleakage in the screw-retained implant–abutment connections of the current study is in accordance with the findings of Scarano et al. They have established that microleakage with screw-retained implant–abutment connections were more than cement-retained implant–abutment connections.
D'Ercole et al. used screws to connect the narrow diameter abutments with wider diameter implants to check the bacterial leakage at the implant–abutment interface. They have observed that screw-retained abutment connections present more bacterial leakage which coincides with the present study results.
Harder et al. evaluated leakage at implant–abutment interface of screw-retained conical connection and screw-retained standard straight connections using endotoxin penetration method. They have detected the presence of endotoxin penetration with both the screw-retained implant–abutment connections.
Berberi et al. studied leakage between the implant–abutment connections among three implant systems using rhodamine B dye penetration. All the samples used in their study were screw retained. They have figured out the presence of the leakage of rhodamine B dye in all the three implant systems.
Sahin and Ayyildiz studied the complication of microleakage on screw-retained implant–abutment connections. They have observed that microleakage can provoke screw loosening and that can be appreciated by reduced removing torque values.
The present study analyzed the microbial contamination on abutment as well as implant surfaces, which is similar to the study conducted by Quirynen et al. Quirynen et al. performed anin vitro study using Branemark implant system to evaluate their resistance against bacterial penetration at implant–abutment interface. They assessed the microbial contamination on the abutment and the implant surfaces. They have detected the microbial contamination on the abutments as well as the implants that is in accordance with the present study results.
Even though the platform-switched implants are scientifically proven to prevent crestal bone loss by shifting the stress concentration away from the crestal bone, the researches on microgap and microleakage in the preservation of the crestal bone loss are less. Hence, this study used platform- switched implants and abutments with screw-retained internal hexagonal connections to evaluate the microleakage at the implant–abutment interface. The results of this present study showed the presence of bacterial contamination on the walls of the implant well, deepest portion of the implant well and the abutment surface. This is due to the microleakage at the implant–abutment interface. The microbial contamination in the present study with screw-retained internal hexagonal connections has found to be more on the walls of the wells of the implants than the deepest portion of the wells of the implants. This may be due to the largest mean microgap with flat to flat internal connections and the microgap decreases exponentially from the outer to inner region of internal connections.
Further researches with various internal connection designs using other modes of retention with different closing torque in platform-switched implants should be conducted to check for their promising results in prevention of microleakage.
- The microgap at the implant–abutment interface is inevitable
- The microleakage at the implant–abutment interface is inescapable
- Microleakage is one of the risk factor for peri-implantitis
- Microleakage may cause screw loosening in screw retained implant–abutment connections.
Limitations of the study
- Intraoral clinical situation is not considered
- Dynamic loading is not considered.
Scope for future studies
- Clinical studies can be done on microleakage at implant–abutment interface
- The surface roughness of implant well can be compared with the abutment surface roughness
- The presentin vitro study can be designed to include dynamic loading
- Effect of various sealants in reducing or eliminating microleakage at implant–abutment interface
- Effect of various closing torque values on microleakage at the implant–abutment interface
- Effect of various geometries of implant–abutment connections on microleakage at implant–abutment interface.
| Conclusion|| |
Within the limitations of the study, the following conclusions were made:
- Platform switched implants with internal hexagonal connections and screw retained abutments presented microleakage at implant-abutment interface.
- Microleakage at the implant-abutment interface led to the microbial contamination in the implant wells and on the abutment surfaces.
- Least amount of microbial contamination was present on the abutment surface. The walls of the wells of the implants had the highest microbial contamination.
Finally, it was concluded that, microleakage is present in the platform switched implants with screw retained internal hexagonal connections at the implant-abutment interface.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23], [Figure 24], [Figure 25], [Figure 26]
[Table 1], [Table 2], [Table 3], [Table 4]