Microbiota Associated with Osseointegrated Implants Restored with Sloping Shoulder and Platform Switched Implants

Introduction: The implant abutment connection is an important factor regarding peri-implant bone remodeling as the highest number of inflammatory cells has been observed at the implantabutment interface. Supracrestal implant position favours the establishment of biological width at the crest sweeping away the microgap and bacterial contamination at the bone crest thereby Original Research Article Markose et al.; BJMMR, 18(1): 1-9, 2016; Article no.BJMMR.29111 2 reduces inflammatory infiltrate. When supracrestal implant installation is done absence of microgap at the bone crest level and reduced inflammatory peri implant cells with minimal bone loss is seen. Among the parameters that can influence crestal bone levels around implants restored with sloping shoulder and platform switching, the composition of the submucosal peri-implant microbiota has been seldom investigated. Therefore, the aim of the present study is to evaluate the bacterial colonization in dental implants inserted in crestal or supracrestal position and correlated it to radiographic measurements of bone remodeling. Materials and Methods: A prospective, randomized parallel clinical trial with 24 subjects who required single-tooth rehabilitations were enrolled in the Group 1 which included 12 subjects with sloping shoulder implants placed 2 mm subcrestal level & Group 2 included 12 subjects with platform switched implants placed crestal level. Radiographic examination was performed at baseline (implant installation) and after 6 months. Clinical and microbiological data were collected after 6 months. Digital radiography was used to assess bone remodeling (marginal bone loss and optical alveolar density). Bacterial profile was analyzed by checkerboard DNADNA hybridization, including a panel of 40 bacterial species. Results: We found higher counts of P. gingivalis, T. denticola, T. forsythia (red complex) at crestal level when compared to subcrestal position and it was least at tooth site. The values of bone remodeling at baseline and after 6 months was statistically significant (p < 0.05) in both the groups, also there was statistically significant difference in bone levels at T2 between crestal and subcrestal groups. Conclusion: The present study results shows that sloping shoulder design with subcrestal implant insertion had significantly less red bacterial complex and bone loss at 6 months post insertion. Platform switched implants showed significantly higher red complex and bone loss.


INTRODUCTION
Biofilm is described as relatively undefinable microbial community associated with tooth surface or any hard nonshedding material. Biofilms are ubiquitous and they form on virtually all surfaces immersed in natural aqueous environment, e.g., water pipes, living tissue, tooth surface, implanted medical devices, dental implants [1].
Osseointegrated dental implants to replace lost teeth in edentulous and partially edentulous patients have become a predictable treatment modality in prosthetic dentistry. Favourable longterm results of dental implant systems have been reported [2]. The success of the prosthetic treatment is widely affected by a various factors which can change the bio mechanical coupling between implant and bone, such as implant location, mechanical and morphological properties of bone, mechanical and geometrical features of implant, and type and magnitude of the load transferred by the implant to the bone, as well as by host factors such as smoking and bacterial environment [3].
The implant abutment connection is considered to be an important factor regarding peri-implant bone remodeling, as the highest number of inflammatory cells has been observed at the implant-abutment interface [4].
In addition to successful treatment, low amounts of plaque and low levels of marginal inflammation have been identified at the implants [5]. Despite the predictable treatment results, with most failures occur during initial healing and the first year of loading, complications do arise during maintenance and retention of implants. The tissues supporting osseointegrated dental implants are susceptible to disease that may lead to implant loss [5].
Sloping shoulder concept was introduced in 1985 with an unique characteristic that facilitates appropriate transfer of occlusal loads to the bone when positioned below the bony crest and it provides room for the bone over the implant which provides support for the inter dental papillae enabling esthetic gingival contours to be easily and consistently achieved. It also provides sensible biological width and provides impressive bone maintenance [6].
The platform switching concept is based on the use of an abutment smaller than the implant neck resulting in a horizontal offset at the top of the implant that separates the crestal bone and the connective tissue from the interface. The biomechanical rationale proposed that by platform switching the stress-concentration zone (from the forces of occlusal loading) is directed from the crestal bone-implant interface to the axis of the implant and so reduces the stress level in the cervical bone area [7].
At the time of implant placement, the implant surfaces are devoid of any local microflora. Soon after the implant insertion the colonization of implant supported restorations leads to increase in peri implant infection, altering the habitat. Microflora adjacent to implant and implant site is influenced by local environment at the interface between peri implant mucosa and implant surface [8].
Many clinical studies have been evaluated about the peri implant microbial composition and few studies have shown that an increase in proportions of spirochetes and motile organisms is associated with increase in probing depth around the implants [9,10]. Studies have also found increased periodontal pathogens around implants with marginal bone loss [11,12].
Supracrestal implant position favours the establishment of biological width at the crest and sweep away the microgap and bacterial contamination from the bone crest and reduces inflammatory infiltrate. Studies have shown that when supracrestal implant installation is done absence of microgap at the bone crest level and reduced inflammatory peri implant cells with minimal bone loss [13,14].
Among the parameters that can influence crestal bone levels around implants restored with sloping shoulder and platform switching, the composition of the submucosal peri-implant microbiota has been seldom investigated. Therefore, the aim of the present study is to evaluate the bacterial colonization in dental implants inserted in crestal or Subcrestal position and to assess the changes in the bone remodeling at baseline and 6 months.

Objectives of the Study
 To evaluate the bacterial colonization in dental implants inserted in crestal and subcrestal position.  To assess the bone remodelling at baseline and 6 months at crestal and subcrestal implant position.

Study Population and Study Design
The population was composed of 24 subjects selected from the OPD of private clinics of Dubai.

Design
Prospective randomized parallel clinical trial.

Sampling Method
Convenience sampling technique.
 Subjects were selected as and when they visited the clinic fulfilling the selection criteria.

Duration of Study
The study was conducted for a period of six months.

Study Procedure
This study was a prospective, randomized parallel clinical trial. 24 subjects who required single-tooth rehabilitations were enrolled at the clinic. All subjects received detailed information about the study and provided written informed consent before the start of the treatment.
In brief, 24 healthy adult subjects with a single missing tooth were randomly allocated to one of the following treatment groups.
Randomization protocol was introduced from a computer generated list to distribute the subjects into 2 groups. Treatment assignments were stored in sealed envelopes for each subject and the envelopes was opened at the time of procedure. Biotech implants (platform switched 4.2 diameter & 8 mm length), Drive implants Sloping shoulder (4.2 mm diameter & 8 mm length) implants was placed in all the subjects.
Patients were evaluated at baseline (T1) and after 6 months (T2). In T1 radiographic examination was performed. In T2 radiographic, and microbiologic data were gathered.

Implant Treatment
The implants (Drive and Bicon) were used in the current study with diameters of 4.2 mm and length of 8 mm. The implant size was selected based on existing bone dimensions. Immediately after local anesthesia crestal incisions was used and full-thickness flaps was elevated to expose the bone. The recipient sites were enlarged according to the protocol of the manufacturer.
Osteotomy procedure was carried out using 10 mm drills with stopper. Subsequent to osteotomy preparation, implants of 8 mm depth was placed in the prepared site. In group 1 dental implants was placed in the edentulous segments with 2 mm below the buccal aspect of the alveolar ridge and group II implants was placed crestally and the flaps were closed with interrupted sutures. Patient was advised to follow standard postoperative instruction, which included ice packs, soft high nutrient diet, postoperative medications were prescribed.
Patients were instructed not to brush the surgical site, but rather to rinse with 2% chlorhexidine gluconate. After about 7-10 days, sutures were removed. After 3 months of surgical procedure, second-stage surgery was initiated, standard gingival former was placed under normal surgical protocol. The patients were then followed-up postoperatively after 2 weeks and closed tray impression technique was taken with additional silicone rubber based impression. Care was taken to avoid any soft tissue injury during the impression procedure. Followed by zirconia crown was delivered to every subject, occlusion was verified and all discrepancies were removed. Post treatment oral hygiene instructions and follow up check ups was provided.

Radiographic Evaluation
Radiographic assessment was performed in the mesial and distal sites of each implant. Periapical radiographies were obtained at baseline and after 6 months, by paralleling technique, The Xray device was calibrated with 70 kV and 7 mA. The exposure time was 0.077 for all implants. Two radiographic parameters were assessed: linear bone loss and optical alveolar density.
All assessments were performed by the same examiner. Intra-examiner concordances were 93.7% for linear measurements within 90.1 mm and 99% for optical density assessments within 93 pixels. Linear measurements corresponded to the distance from the crest of the bone to the base of the implant. and were obtained in millimeters. The bone regions of interest (ROIs), approaching 1 mm 2 , were positioned laterally to each implant, at the mesial and distal most coronal point of the implant/bone contact, in the alveolar bone crest (without touching the implant). For the ROIs confection, a radiodense net with 1 mm 2 was positioned above the digital sensor and was used as a calibrator to the X-ray system. Optical alveolar density was determined by the average intensity of the grayscale in a diagonal line from the left inferior vertex to the right superior vertex of the ROI. The grayscale varied from 0 to 256 pixels, where 0 corresponded to black and 256 to white [15].

Statistical Tests
The data was tabulated in microsoft excel sheet (2007), ANOVA was used to compare the bacterial complexes in dental implants inserted in crestal and subcrestal position. Tukey's Post Hoc analysis was performed for intragroup comparison.
Bone remodelling in baseline and after 6 months was assessed using student ' t' test.

RESULTS
From the 24 patients who were included, 22 subjects were assessed.

Microbiological Profile
In the present study 40 microbial species were examined. The mean values of microbial species at the tooth site, subcrestal and the crestal level are presented in the Fig. 1.

Bacterial Complexes
Difference in the mean bacterial count in green, orange and red complexes of crestal, tooth site and subcrestal groups was analysed using one way ANOVA. There was a statistically significant difference in the bacterial counts of red complex only(p -< 0.05), further Tukey,s post hoc analysis for intragroup comparison in red complex showed that there was statistically significant difference between crestal and subcrestal, crestal and tooth site groups (p < 0.05) ( Table 1).

Statistical Tests
The data was tabulated in microsoft excel sheet (2007), ANOVA was used to compare the bacterial complexes in dental implants inserted in crestal and subcrestal position. Tukey's Post Hoc analysis was performed for intragroup comparison.
Bone remodelling in baseline and after 6 months was assessed using student ' t' test.

RESULTS
From the 24 patients who were included, 22 subjects were assessed.

Microbiological Profile
In the present study 40 microbial species were examined. The mean values of microbial species at the tooth site, subcrestal and the crestal level are presented in the Fig. 1.

Bacterial Complexes
Difference in the mean bacterial count in green, orange and red complexes of crestal, tooth site and subcrestal groups was analysed using one way ANOVA. There was a statistically significant difference in the bacterial counts of red complex only(p -< 0.05), further Tukey,s post hoc analysis for intragroup comparison in red complex showed that there was statistically significant difference between crestal and subcrestal, crestal and tooth site groups (p < 0.05) ( Table 1).

Statistical Tests
The data was tabulated in microsoft excel sheet (2007), ANOVA was used to compare the bacterial complexes in dental implants inserted in crestal and subcrestal position. Tukey's Post Hoc analysis was performed for intragroup comparison.
Bone remodelling in baseline and after 6 months was assessed using student ' t' test.

RESULTS
From the 24 patients who were included, 22 subjects were assessed.

Microbiological Profile
In the present study 40 microbial species were examined. The mean values of microbial species at the tooth site, subcrestal and the crestal level are presented in the Fig. 1.

Bacterial Complexes
Difference in the mean bacterial count in green, orange and red complexes of crestal, tooth site and subcrestal groups was analysed using one way ANOVA. There was a statistically significant difference in the bacterial counts of red complex only(p -< 0.05), further Tukey,s post hoc analysis for intragroup comparison in red complex showed that there was statistically significant difference between crestal and subcrestal, crestal and tooth site groups (p < 0.05) ( Table 1).  t -test was applied to analyse the difference between the means of the groups *-P value <0.001 (i.e. highly significant difference between the sub-crestal and crestal group)

Radiographic Bone Analysis
Bone loss and optical alveolar density at T1 (Baseline) and T2(6 Months) in subcrestal and crestal are presented in Table 2 above. There was a slight change in optical alveolar density from T1 -T2 in both the groups, but the difference was not statistically significant (p > 0.05). The values of bone remodeling at baseline and after 6 months were subjected to paired 't' test, it was seen that there was statistically significant bone loss (p < 0.05) in both the groups, also there was statistically significant difference in bone levels at T2 between crestal and subcrestal groups.

DISCUSSION
The Dental implant success rates showsthat most of the implant failure following insertion and after attachment of implant abutment is attributed to factors like surgical trauma during insertion, occlusal overload, supporting bone quality, habits and location of microgap between implant abutment interface [8].
There is a disagreement regarding implant insertion level and its relation to bone crest and its influence on periimplant bone remodeling. Thus, this study was performed to evaluate the effect of the implant insertion level on bacterial profile and peri-implant bone remodeling.
We found higher counts of P. gingivalis, T. denticola, T. forsythia (red complex) at crestal level whwn compared to subcrestal position and it was least at tooth site. There was a statistically significant difference in the bacterial counts of red complex. The findings are contradictory to studies conducted by Canullo et al. [8] Mariano Sego et al. [15] this could be due to tooth selection and implant design and positioning. Canullo et al. [8] study was done in completely edentulous patients whereas the present study was done in partially edentulous patients. In mariano et al. [15] study supracrestal positioning of implant was done whereas this study subcrestal position was done.
There was a slight change in optical alveolar density from T1 -T2 in both the groups, but the difference was not statistically significant (p > 0.05). The values of bone remodeling at baseline and after 6 months were subjected to paired 't' test, it was seen that there was statistically significant bone loss (p < 0.05) in both the groups, also there was statistically significant difference in bone levels at T2 between crestal and subcrestal groups. The changes in reduced bacterial complex and bone loss is due to the implant design and subcrestal positioning.
The present study used sloping shoulder (Bicon) implants were used. Sloping shoulder (Bicon Implants) is a screwless implant system. The implant and implant-abutment unit connect by means of a 3.0°locking taper. Assembly is achieved by tapping the abutment into then matching socket in the implant. A high clamping force between abutment and implant is generated through elastic deformation of both parts. During engagement, the high friction force resulting from the relative slip between the friction surfaces yields high contact forces. This interaction results in the surface oxide layers breaking down and the asperities fusing, commonly referred to as cold welding. The locking-taper connection provides a frictional bacterial seal with excellent clinical reliability [16] Bicon has demonstrated the ability of their connection to provide an adequate microbial seal. The cold weld formed between the implant and abutment has been shown to create a hermetic seal keeping bacteria from colonizing the implant. Because there is no retaining screw with the Bicon system, there are no concerns about screw loosening. Generally in the posterior part of the mouth, occlusal forces decrease the preload of the retaining screw, but with the Bicon implant the occlusal forces strengthen the connection between the implant and abutment [17].
Meanwhile, other studies done by Buser etal and Huang et al. have found advantages with subcrestal placement of dental implants [18,19]. study found statistically significant difference when bone remodeling was evaluated in twopiece implants inserted 2 mm subcrestally and at the bone level. This result is in contradiction with studies by Heijdenrijik et al. and Todescan et al. which found no effect of the microgap location in bone remodeling [20,21,22]. Several factors may account for the conflicting results, as interface implant/abutment and healing time. In our study, the interface implant/abutment in the subcrestal group was located 2 mm. Boynueǧri et al. [23] have located the microgap 2.8 mm above the bone crest. Piattelli et al. [24] located the interface implant/ abutment 1 -2mm above the bone crest and found that this position was favorable to a minor bone loss in this group. However, Guruprasada et al. [25] and Piattelli et al. [24] found no significant differences in peri-implant bone remodeling comparing conventional loading and immediate loading protocols.
A reduced sample and short term follow up for bone remodeling is limitation of our study. Studies with greater sample sizes and longer follow-ups are needed to investigate if these results and their clinical implications will be maintained.

CONCLUSION
The present study results shows that sloping shoulder design with subcrestal implant insertion had significantly less red bacterial complex and bone loss at 6 months post insertion. Platform switched implants showed significantly higher red complex and bone loss.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.