Comparison of bacterial colonization of implant abutment internal recess in internal hex and conical connection implants: An in vivo prospective study

Abstract

Aim:

Microbial assessment of internal recess of two different internal implant connections in an in vivo study.

Study Settings and Design:

This randomized, in vivo study included 40 patients requiring mandibular single-tooth implant-supported prostheses, allocated into two equal groups based on implant connection design.

Materials and Methods:

Group Internal Hex (IH) and group Conical Connection (CC) received implants with IH and CC, respectively. On the day of prosthetic loading, peri-implant sulcular fluid (PISF) sample was collected and stored. PISF samples were recollected after 3 months. Subsequently, the screw access hole was exposed, and the abutment screw was removed. Implant-abutment recess was washed with saline, lavage was sent for microbiological assay. The abutment screw and crown were restored. PISF was analyzed for interleukin-6 (IL-6).

Statistical Analysis Used:

For nonparametric values Chi square test and for parametric values t-test was used to analyse the data.

Results:

No implant failure or patient fallout was observed. In Group IH, 17 out of 20 samples were positive for aerobic viz-a-viz 7 samples from Group CC; P = 0.001. For anaerobic bacteria, similar results were obtained with a number of positive samples 19 as compared to 6 in respective groups; P = 0.00002. IL-6 values did not differ significantly from baseline to 3 months in either group.

Conclusion:

Within the limitations of the study, results show higher bacterial contamination of implant recess in IH than CC. However, the bacterial load had an insignificant contribution to IL-6 levels in PISF of the patients of either group in this time period.

INTRODUCTION

For the past few decades, titanium dental implants have been considered an effective treatment of choice for oral rehabilitation. Failure of this treatment is attributed to various mechanical or biological causes which result in excessive peri-implant bone loss. Various suppositions have been explored and reported to explain this loss, implant–abutment interface microgap being one of the plausible causes.[1]

Two-piece implants have a hollow recess in the implant and a micro gap invariably exists between the implant and abutment. Cyclic loading during occlusion and micro-movement at Implant abutment junction (IAJ), causes ingress of oral fluids, metabolic substrates, and microbial contaminants into the implant internal recess, facilitating the proliferation of the bacteria. This bacterial reservoir serves as a source for microleakage from the IAJ.[2] The design of the implant–abutment connection (IAC) can markedly influence the degree of bacterial leakage and the resulting peri-implant inflammation.[3] External hex connections have many disadvantages, prompting the introduction of internal connections.[4] Internal connections are mainly of two types; passive (internal hex [IH]) connection and conical hex or morse taper interfaces. The IH connection is a slip-fit joint characterized by a microgap of approximately 20–25 µm, on the other hand conical tapered connection features a tube-in-tube friction-fit design with a much narrower gap of about 2–3 µm and a cold-welding effect, which can help delay bacterial infiltration. Literature extensively supports the use of internal connection, particularly conical over external ones.[4,5] However, comparisons between results of the use of IH versus conical hex connections are limited and that too with diverse implant and patient-related parameters,[6,7] and no study has definitively confirmed the superiority of any of these connections. Grandi et al. in their 5-year follow-up and Mihali et al. in their 3-year follow-up revealed no measurable or clinically relevant differences in marginal bone loss between the two types of IACs.[1,8] Many in vitro studies have reported increased bacterial load in other connections viz-a-viz morse taper one.[9,10,11,12,13] However, in in vivo conditions, Canullo et al. have reported decreased bacterial load in conical connection (CC) as compared to internal and external hex connections.[14]

Microleakage at IAJ may lead to inflammation in the surrounding tissues and subsequent bone destruction depending upon bacterial load and type. Inflammatory infiltration can elevate the levels of various biomarkers in peri-implant sulcular fluid (PISF). Interleukin-6 (IL-6) is one such biomarker which remarkably increases in the presence of inflammation.[15] Insufficient and contradictory in vivo and in vitro literature on microleakage at the IAJ, particularly with respect to internal IAC, prompted the present in vivo investigation to assess the microbial load at the implant–abutment interface in the two most commonly used IAC, i.e. IH and Conical hex connection. Levels of IL-6 were analyzed in PISF of all patients to assess the host tissue response to this microbial leakage, during initial phase of osteointegration. The null hypothesis formulated was that there would be no difference in the extent of bacterial load from the internal recess of the two connections.

MATERIALS AND METHODS

Study subjects for the present longitudinal study were selected from patients reporting to the department of prosthodontics of the institute for dental implants. Forty patients requiring single tooth implant-supported prosthesis, with a minimum residual bone height of 10 mm in the posterior region of the mandible were enrolled for the investigation. None of the included patients had removable partial or complete dentures opposing the implant site. The participant group comprised 24 males and 16 females. The age distribution spanned 22–78 years for males and 23–74 years for females. Alcoholics and smokers were excluded. Other exclusion criteria were patients with poor oral hygiene, with untreated periodontitis, uncontrolled diabetes mellitus, or patients with any contraindication to implant surgery. Patients with D4 bone quality, assessed on the basis of tactile sensation, during implant bed preparation were excluded, and the same number of new patients were included in the study.[16] The sample size for the study was calculated by taking a confidence interval of 80% with a z score of 1.28. The sample size came to be 40.96. For convenience of grouping, 40 patients were included in the study.

Before enrollment, all participants were thoroughly informed about the study, and written consent was obtained. The study was given ethical clearance by the Institute Ethics Committee of the University vide letter no. PUIEC/2018/127/A-1129/10. Implants had different prosthetic connections but the same macro design, to eliminate its influence on the study results. Both implant systems had tapered body. For IH, Bio horizon implant system was used and for conical hex, Megagen, AnyRidge hex connection with 5° taper was used [Figure 1].

Figure 1.

Different implant connections used (a) Biohorizon(internal hex connection) (b) Megagen (internal conical connection) Image source: https://imegagen.com, https://www.biohorizons.com

After inclusion, the patients were clinically assessed for the size of the implant to be given. The participants were randomly distributed into two groups using computer-generated numbers using web-based system, random.org, which were enclosed in sealed envelopes. The allocation sequence was generated by an independent researcher. The envelopes were opened by the clinician just before the implant surgery. The patient was advised to have 625 mg amoxiclav, 2 h before surgery, followed by the same dose along with dispersible piroxicam tablets twice daily for 5 days. Group CC was given implants with a conical hex connection and Group IH was given implants with IH connection. The implant placement site was prepared using nonguided drilling protocol using sequential drills [Figure 2]. All implants were placed using a submerged technique and delayed loading protocol [Figure 3].

Figure 2.

(a) Full thickness incision at midcestal position, (b) Sequential drills for preparing osteotomy site

Figure 3.

(a) Tapered implant screwed in bone using torque wrench, (b) Gingival former at implant site, (c) Implant level impression with implant analog

Prosthetic phase and sample collection

After 12 weeks, 2^nd stage surgery was performed by replacing the cover screw with a healing abutment. On the next visit after 10 days, the healing abutment was unscrewed, followed by disinfection of implant recess with 0.2% chlorhexidine (CHX). Impression was taken using closed tray impression technique for most of the patients, for prosthetic fabrication. Pre autoclaved new healing abutment was placed on the implant. On the next recall, after the removal of healing abutment, the screw access hole was disinfected with 0.2% CHX and air-dried. A precemented metal ceramic crown, on stock abutment with screw access hole (disinfected with 0.2% CHX), was loaded on the implant with abutment screw tightened in accordance with the manufacturer’s guidelines. The screw access hole was disinfected with 0.2% CHX and plugged with PTFE sealed with 2 mm of composite restoration. All the aseptic procedures followed in the study were in accordance with Schoenbaum et al.[17] After the crown placement, for PISF collection, the implant site was carefully dried and isolated with cotton rolls to minimize salivary contamination. 2 µl of PISF was collected using 1–5 µl calibrated Hirschmann’s microcapillary pipette (Sigma-Aldrich, St Louis, MO, USA). The pipette was gently placed, extracrevicularly, at the gingival margin for about 5–8 min for collection of sample [Figure 4a]. Contaminated samples, whether with blood or saliva, were not considered for further evaluation.[18] Reattempt was made half an hour later or the next day as per patient’s convenience. The PISF was transferred to 2 ml labeled storage vial containing 198 μl of normal saline as a standard diluent. The vials were stored at −80ºC till the day of analysis.

Figure 4.

(a) Peri-implant sulcular fluid collection, (b) Inoculation of different samples

To ensure optimal oral hygiene, patients were instructed to brush their teeth twice a day and use a 0.2% CHX mouthwash daily. The patients were recalled after 3 months. After appropriate isolation, 2 μl of PISF was collected with a micropipette from the implant site and stored as before. The screw access hole was subsequently cleared of the composite restoration. The abutment screw was removed and the implant fixture recess was washed with premeasured quantity of saline (200 µl) and aspirated (lavage) using a sterile autopipette and sent for microbiologic analysis on the same day, in a labeled container. The abutment recess was again disinfected with 0.2% CHX and the abutment placed with a crown was re-torqued to 35N and sealed. No case of implant failure was observed and no patient dropped out of the study. After this visit, the patient was advised 6 monthly follow-up for assessment of probing depth and marginal bone levels.

Microbiological and biochemical analysis

For microbial analysis, lavage samples were plated on blood agar and MacConkey agar [Figure 4b]. To isolate aerobic organisms, one blood agar and one MacConkey agar plate were incubated at 37°C for 24 h. For anaerobic culture, a blood agar plate was placed in a McIntosh jar with a gas pack and incubated at 37°C for 48 h. After incubation, the plates were examined for bacterial colony growth using a colony counter. The predominant colony type from each sample was identified based on Gram staining and colony morphology. Gram-positive and Gram-negative bacteria were distinguished by microscopic examination of the slides under oil immersion (×100).

The IL-6 protein level in PISF was determined by IL-6 specific sandwich ELISA system. PISF samples were thawed only on the day of analysis. The Ray Bio human IL-6 ELISA kit was used to quantify human IL-6 in PISF as per manufacturer’s instructions. Patient samples were run in duplicate and the average was taken as IL-6 levels of each patient. The concentration obtained on the reader was multiplied by the appropriate dilution factor and a final concentration of IL-6 of patients was calculated for both baseline and 3-month PISF samples. The sensitivity of the kit was 3 pg/ml and the detection range was 3–1000 pg/ml. The results of the study were subjected to suitable statistical analysis using SPSS software version 22.0 (IBM SPSS Statistics Inc., Chicago, Illinois, USA). Kolmogorov–Smirnov Z-test ensured the normality of the data. The positivity of the samples was compared using nonparametric Chi-square test. Parametric t-test was conducted for comparing number of colonies from each connection and to compare IL-6 values of the PISF samples in both groups.

RESULTS

Microbial contamination of lavage from implant internal recess was assessed in two different IAC (20 each); IH and conical hex implant abutment connection. Group IH demonstrated significantly higher infiltration of aerobic (85%) as well as anaerobic (95%) microorganisms than Group Conical hex connection (Group CC). Colony count for both types of organisms was also significantly higher in Group IH [Table 1].

Table 1.

Presence of aerobic/anaerobic micro-organisms and colony count in wash from implant internal recess of the two groups

IAC	Number of samples	Aerobic			Anaerobic
		Positive samples	Colony count	Mean±SD	Positive samples	Colony count	Mean±SD
Group IH	20	17	200	10±14.367	19	23	1.15±0.489
Group CC	20	7	32	1.6±3.101	6	8	0.4±0.680
P		0.001*	0.015*		0.00002*	0.0002*

*P<0.005, Statistically significant. SD: Standard deviation, Group IH: Group internal hex, Group CC: Group conical hex connection, IAC: Implant-abutment connection

Biochemical analysis of IL-6 in PISF around the two IAC systems at baseline (implant loading) and at 3 months postloading showed no significant differences from baseline values in either group. The mean of IL-6 levels in 20 PISF samples from Group IH at the time of loading was 286.50 ± 50.9 pg/ml and 268.30 ± 64.5 pg/ml in Group CC. After 3 months of loading, mean IL-6 values were 264 ± 71.2 pg/ml and 247.06 ± 49.6 pg/ml in both groups, respectively [Table 2].

Table 2.

Comparison of interleukin-6 values in peri-implant sulcular fluid in the two groups from baseline to 3 months

Groups	Number of samples	Baseline IL-6 levels, mean±SD (pg/mL)	3 months IL-6 levels, mean±SD (pg/mL)	P
Group IH	20	286.50±50.9	264±71.2	0.793*
Group CC	20	268.30±64.5	247.06±49.6	0.561*

*P<0.005, Statistically significant. SD: Standard deviation, IL-6: Interleukin-6, Group IH: Group internal hex, Group CC: Group conical hex connection

DISCUSSION

Microbial leakage to and from implant internal recess at the IAJ has been implicated as one of the primary causes of peri-implantitis. In the current investigation, a significant difference in bacterial load was observed in the microbial analysis of wash samples collected from the internal recess of the IH connection compared to the conical hex connection. This finding led to the rejection of the null hypothesis, which stated that there is no difference in bacterial load at the implant–abutment interface between the two connection types.

A maximum number of samples of recess wash from IH connection have shown the presence of microorganisms including both Gram-positive (G+ve) and Gram-negative (G-ve) aerobes and anaerobes. Significantly higher colony count also signifies a higher concentration of bacteria in the recess and subsequent more chances of microleakage to peri-implant area. The CCs do show bacterial colonization but to a significantly lesser extent. These findings are consistent with the in vivo study conducted by Canullo et al. who reported lower bacterial load at the IAC in internal CC followed by IH and external hex connection implants, although the results were statistically significant for Treponema denticola and Porphyromonas gingivalis bacterial species (red-complex bacteria), both of which are major periodontitis causing pathogens.[14] In an in vitro study, Tripodi et al. investigated the outward movement of Pseudomonas aeruguenosa and Actinobacillus actinomycetemcomitans from the internal recess of conical taper and IH implant to the peri-implant sulcus, with results in favor of the former connection.[12] Khorshidi et al. also reported an outward movement of Streptococcus mutans from the internal recess of 70% of butt joint connections as compared to 10% of morse taper connections.[13] Sahin and Ayyildiz, in their in vitro study, further demonstrated that IH IAC showed higher microleakage as compared to implants with morse taper connections.[9] However, Gherlone et al. and Ardakani et al. in their in vitro studies compared double conical, morse taper, conical, and IH IAC for microleakage and reported an insignificant difference in microleakage from IH and conical IAC.[10,11]

Cytokines and enzymes released from local host tissue in response to bacterial invasion may lead to the degradation of connective tissue collagen and alveolar bone. PISF is an important source of inflammatory markers that signal any change around the implant site. IL-6 was evaluated in PISF of both types of connection at baseline and 3 months after crown placement to study any increase in its levels due to microleakage from IAJ. Levels of the marker were statistically indifferent in both the groups from baseline to 3 months; rather there was a slight but statistically insignificant decrease in the levels of the marker. This can be attributed to the fact that though microleakage does occur, bacterial contamination of peri-implant area was insufficient to elevate IL-6 to significantly higher levels in PISF around study implants. The findings of the present study are also partially in agreement with those reported by Canullo et al. who reported insignificant differences in the percentage of positive sites in PISF obtained from implant sites in various studied connections with differences tending to significance for CC. Furthermore, statistically nonsignificant decrease in bacterial load of red complex bacteria was found in CC versus other connections, 5-year postimplant placement.[14]

CONCLUSION

The data obtained in the current study indicate a strong predilection of IH connection implants as harboring sites for the growth of both aerobic and anaerobic microorganisms at IAJ as compared to CC implants. Within 3 months of implant placement, these bacterial reservoirs did not significantly raise IL-6 in PISF in either of the connections. However, the design of the current study restricts the conclusions that can be made. The main limitation of the study was the short duration of the follow-up. To confirm the findings of this study, future research should focus on expanding the sample size, lengthening the follow-up period, and performing a qualitative analysis of bacterial load in the collected samples. This will further help to better understand the clinical significance of the differences in the bacterial load in various implant connections and their causal relationship in the evolution of peri-implant disease.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Panjab University, PURSE grant, DST, New Delhi.