Interleukin-1β level in peri-implant crevicular fluid and its correlation with the clinical and radiographic parameters.

BACKGROUND AND OBJECTIVES: Assessing only the clinical and radiographic parameters for evaluation of dental implants may not be enough as they often reflect extensive inflammatory changes in the periodontal tissues. As peri-implant crevicular fluid (PICF) can give us a more prompt and objective measure of the disease activity, the purpose of this case series is to assess the peri-implant health status of single tooth dental implants not only clinically and radiographically but also biochemically.
MATERIALS AND METHODS: Thirteen patients were subjected to dental implants at single edentulous sites using a conventional surgical approach. At baseline, 6 months, and 12 months after implant placement, the clinical and radiographic parameters were recorded. Additionally, IL-1β in PICF was estimated using the ELISA kit at 6(th) and 12(th) month.
RESULTS: The clinical and radiographic parameters differed significantly around the implants at different time intervals with IL-1β levels showing highly significant differences between 6 months (31.79 ± 12.26 pg/μl) and 12 months (113.09 ± 51.11 pg/μl). However, Spearman's correlation coefficient showed no correlation with the clinical and radiographic parameters. INTERPRETATION AND
CONCLUSION: Assessment of the various parameters confirmed that all the implants had a healthy peri-implant status. Although the levels of IL-1β in PICF were elevated at the 12(th) month, they were well within the healthy range as observed by previous studies. This indicates that IL-1β, a biochemical marker, can be used as an adjunct to clinical and radiographic parameters in the assessment of EARLY inflammatory changes around implants.

INTRODUCTION

The use of endosseous implants to treat completely and partially edentulous patients has become a standard of care in dentistry. However, many of the implants may succumb to an aggressive inflammation involving the hard and soft tissues surrounding the implant showing signs of bleeding on probing, purulence, pocket formation, and bone loss.[1] This inflammation is termed as peri-implantitis.[2]

As the criteria for evaluating disease activity around dental implants are mainly based on clinical and radiographic methods, they often reflect extensive inflammatory changes. The tissue appearance may not be a good clinical measure for monitoring early peri-implant health changes.[3] The radiographic evidence of bone loss is also detectable only after a significant demineralization has taken place.[4] Furthermore, the evaluation of clinical and radiographic changes is often subjective. Ideally, the presence of peri-implantitis should be detected objectively and during the early inflammatory phase to minimize the tissue damage and increase the potential for therapeutic success.[3]

Peri-implant crevicular fluid (PICF) is an osmotically mediated inflammatory exudate originating from the vessels of the gingival plexus. It contains host-derived enzymes, inflammatory cytokines, and tissue breakdown products.[5] Based on the fact that periodontitis and peri-implantitis are similar in clinical manifestations and microbial profile, it seems that the IL-1ί stimulated during peri-implantitis may also be the same cytokine that is released during periodontitis and may cause destruction of the supporting peri-implant tissues. Thus, IL-1ί has been established to be an important marker in PICF to evaluate the tissue destruction around dental implants.

Kao et al.[3] reported elevated levels of IL-1β which were three times around the diseased implants as compared to the healthy ones. Similarly, Panagakos and coworkers[6] also observed greater levels of inflammatory cytokines in PICF around early and advanced peri-implantitis groups as compared to the healthy group. As it is difficult to establish a diagnosis of peri-implantitis only on the clinical basis, it becomes logical to have an objective method in the form of PICF analysis to measure the disease activity. Hence, the purpose of this case series was to investigate whether a correlation exists between IL-1ί levels in PICF and the clinical and radiographic parameters used to assess the peri-implant health status at 6 months and 12 months.

MATERIALS AND METHODS

In this prospective case series, 13 patients (nine males and four females) in the age range of 20-61 years (mean age of 39.23 ± 12.65 years) were selected from the Out Patient Department of Periodontology and Implantology, Bapuji Dental College and Hospital, Davangere, from 2010 to 2011. Ethical approval for the study was obtained from the Institutional Ethical Committee. All patients were informed and explained about the nature and course of treatment and an informed consent was obtained from them before starting the treatment.

To be included in the study, the patients had to fulfill the following criteria: (1) age more than 18 years with a missing mandibular posterior tooth. (2) Presence of a single edentulous site with adjacent healthy teeth. (3) Presence of adequate bone volume and vertical inter-arch space to accommodate an implant with prosthesis of appropriate size as determined by clinical inspection, pre-operative radiographs, and CT scan before implant placement. (4) Patient with good oral hygiene. The exclusion criteria included: (1) Medical history that would complicate the outcome of the study. (2) Dental history of bruxism, parafunctional habit, and/or lack of stable posterior occlusion. (3) Habit of smoking or alcohol consumption.

All implants were recorded and evaluated with modified plaque index (mPI),[7] simplified gingival index (sGI),[8] modified sulcular bleeding index (mSBI),[7] presence or absence of infection around the implant, probing depths at four sites (mesial, distal, facial, and palatal) using TPS probe* and implant mobility (according to clinical implant mobility scale[9]) at baseline, 6 months, and 12 months.

Intra-oral periapical (IOPA) radiographs were taken using the long cone paralleling technique and assessed at the time of implant placement, at 6 months, and 12 months. The distance from implant shoulder to the first bone-implant contact was measured on the radiographs using an Image processing system on the computer†. Panoramic radiographs were taken before placement of implant to rule out any gross pathology of the jaws. Computed tomographic scans (C.T.) were done to assess the quantity as well as quality of bone around the intended site, and to determine the size of implant to be placed.

Study design

Thirteen dental implants were placed in single edentulous sites and submerged. After the healing period of 4-5 months, second stage surgeries were performed. After collecting the sixth month PICF sample, fabrication of the prosthesis was undertaken. Finally, after the delivery of the prosthesis, 12th month PICF samples were collected and subjected to biochemical analysis [Figure 1].

Figure 1

Study design

Surgical procedure

The patient was subjected to extra-oral scrubbing with 5% povidone iodine solution and prerinsing with 0.2% chlorhexidine digluconate mouthwash. After achieving adequate local anesthesia, a mid crestal incision was given at the implant site which was extended to the mesial and distal teeth by crevicular incisions. Full thickness buccal and lingual mucoperiosteal flaps were elevated to expose the alveolar bone. Width and quantity of bone was then assessed at the crest of the alveolar ridge. After evaluating the dimensions of the ridge and findings of the radiographs, final decision regarding the dimensions of the implant was made. Surgical template was then used to determine the position of the implant. Drilling of the osteotomy site was done according to the manufacturer's instructions. Implant‡ was then placed in the osteotomy site with the implant-abutment junction at the bone level. After the implant placement, the cover screw was placed over the implant and the flaps were sutured back in place [Figure 2b and c].

Figure 2

(a) Pre-operative occlusal view; (b) Implant placement; (c) End of first stage; (d) Second stage & – Healing cap placement; (e) PICF collection at sixth month; (f) PICF collection at 12th month; (g) Pre-operative radiograph; (h) 6th month radiograph; (i) 12th month radiograph

Patients were prescribed Amoxycillin 500 mg three times daily for 5 days and Diclofenac sodium 50 mg three times daily for 3 days. Patients were advised to rinse with chlorhexidine digluconate (0.2%) for a period of 15 days. One week later, sutures were removed.

Second stage surgery

After a healing period of 4-5 months, incisions were placed over the implant site and soft tissue was reflected sufficiently to allow removal of cover screw. Healing abutments were placed and gingival tissue was sutured around it [Figure 2d]. After 1 week, suture removal was performed and oral hygiene instructions were reinforced. Fabrication of the prosthesis was undertaken after the first PICF sampling recall was over.

Collection of peri-implant crevicular fluid

PICF was collected at the 6th month and 12th month postoperatively [Figure 2e and f]. Prior to the collection of crevicular fluid, the implant was first isolated with cotton rolls and the area was dried. After removing the supragingival plaque, a standardized volume of 3 μl PICF from the implant site in each patient was collected using calibrated, volumetric microcapillary pipettes§ positioned extracrevicularly on the margin of the gingiva and immediately transferred to an eppendorf tube containing phosphate buffered solution and frozen at −70°C.[10] Samples visibly contaminated with blood and saliva were discarded. Assay was then performed using IL-1β ELISA test kitκ according to the manufacturer's instructions.

Statistical analysis

Student's paired t-test or Wilcoxon signed-rank test was used for intra group comparisons whereas relationship between clinical, radiographic parameters, and biochemical parameters was assessed by the Spearman's correlation coefficient. In all the analyses, a P < 0.05 was considered to represent a statistically significant difference.

RESULTS

All patients participated until the end of the follow-up period with no clinical dropouts reported. Thorough clinical and radiographic examinations were carried out at the 6 and 12 months. There was an evidence of infection around one implant in the form of a cover screw abscess at the fifth month post implant insertion. The abscess was drained and second stage surgery performed simultaneously. In the remaining 12 patients, there was no evidence of any infection around the implants during 12 months of the follow-up duration.

Full mouth scores of mPI, sGI, and mSBI showed only marginal changes during 6 and 12 month recall. There were no significant differences between the 6th month and 12th month probing depth values at the midbuccal and lingual sites. However, for the mesial and distal sites, significant differences were observed Table 1 and Graph 1].

Table 1

Probing depth

Graph 1

Mean difference in probing depth at different intervals

The radiographic analysis using Image J software revealed that there was a statistically significant difference in the crestal bone levels between baseline, 6 month and 12th month values at both mesial and distal sides [Table 2 and Graph 2].

Table 2

Radiographic analysis using Image J analysis software from shoulder of implant to the first bone.implant contact on mesial and distal side

Graph 2

Mean bone loss on mesial and distal side of implant at different intervals

Biochemical analysis of IL-1β level revealed mean values of 31.79 ± 12.26 pg/μl and 113.09 ± 51.11 pg/μl at the 6th and 12th month recall, respectively. The mean difference of 81.30 ± 47.08 pg/μl was highly significant [Table 3 and Graph 3]. Interestingly, the case with the cover screw abscess was associated with drastic differences in values of IL-1β at 6 months (232.536 pg/μl) and 12 months (126.213 pg/μl).

Table 3

Mean IL-1β levels in PICF at different intervals

Graph 3

Estimation of IL-1β level in PICF (using ELISA kit)

Finally, Spearman's correlation coefficient revealed no significant correlation between the clinical, radiographic, and biochemical parameters at both 6 and 12 months [Table 4].

Table 4

Correlation of biochemical parameter (IL-1β levels in PICF) with clinical and radiographic parameters

DISCUSSION

Disparities in the results have been seen in different studies while assessing peri-implant mucosal health. Bleeding on probing as an indicator of the level of inflammation may be more directly related to the tightness of the mucosa around the abutment, and probing may result in tissue penetration, with subsequent bleeding occurring at otherwise healthy site. Similarly, mucosal inflammation assessment was difficult, since the peri-implant mucosa was frequently non-keratinized and therefore appeared “redder”. Also, the residual scaring from the surgical procedures of implant placement surgeries added to the difficulties in the assessment. Hence, PICF analysis has been strongly recommended while assessing peri-implant mucosal conditions.[8]

Several biochemical mediators in the gingival crevicular fluid (GCF) around natural teeth have been identified as potential host markers for periodontal disease activity and progression. However, only a few studies have reported the association between signs of peri-implant inflammation and increased levels of inflammatory mediators in the PICF. IL-1β is a multifunctional cytokine with diverse biologic activities implicated in the pathophysiology of not only periodontitis but also peri-implantitis.

In the present case series, as the sGI, mSBI, and mPI showed no major differences at 6 months and 12 months, implied that the oral hygiene maintenance of the patients had not deteriorated over the follow-up period of 1 year. There were no significant differences between the 6th month and 12th month probing depth values at the midbuccal and lingual sites. However, significant differences were seen for the mesial and distal sites. This implies that after delivery of the prosthesis oral hygiene maintenance was much easier at the buccal and lingual surfaces as compared to the mesial and distal surfaces. As the implant shoulders were straight and horizontal in contrast to the scalloped CEJ around natural teeth, this made the implant supported crown margins to be located more submucosally on mesial and distal sides as compared to the buccal and lingual sides.[11] Hence, the patient's oral hygiene efforts must have been more compromised on interproximal sides than facially or lingually.[12]

The radiographic analysis revealed that there was a significant difference in the crestal bone levels between baseline, 6 month, and 12th month values at both mesial and distal sides. These radiographic crestal bone changes can be attributed to the fact that during the first year after implant placement, bone healing and remodeling occurs around the implants.

After completing the 12th month recall, all the 13 implants placed were considered healthy as there was minimal gingival inflammation, no gross radiographic evidence of bone loss, pocket depths were less than 4 mm, no attachment loss greater than 2 mm, and no pain at the implant sites. These criteria for health assessment were also adopted by previous studies of Kao et al.[3] and Panagakos et al.[6]

IL-1β levels were also estimated in the PICF around the implants using an ELISA kit. The low values at the 6th month may be due to the presence of the healing abutment which caused minimal peri-implant mucosal irritation, whereas the high values at the 12th month can be attributed to the submarginal placement of the implant supported crown margins. One more possibility for such high values at the 1 year recall may be due to the effect of occlusal loading of the prosthesis.[13] Never-the-less, both the 6 and 12 month values of IL-1β in peri-implant mucosa were well within the range of healthy implants as seen by Kao et al.,[3] Ataoglu et al.,[14] and Murata et al.[15]

Interestingly, the case with cover screw abscess had high IL-1β level at the sixth month (232.536 pg/μl). The 12th month value of IL-1β for the same case dropped to 126.213 pg/μl. This reduction could be due to drainage of the abscess and simultaneous performance of the second stage surgery which restored the health of the peri-implant mucosa by the 12th month recall. Our findings are consistent with the a case report by Curtis et al.[16] The reversibility of IL-1β levels was also noticed by Salvi et al.,[17] in their experimental peri-implant mucositis study.

The correlation of biochemical parameters with clinical and radiographic parameters using the Spearman's correlation coefficient revealed no significant correlation between the above parameters at both 6 and 12 months. As the clinical and radiographic parameters in the present study were within the healthy range, this could have been the reason for the absence of any significant correlation with the IL-1β levels in the PICF.

Findings of this case series indicates that there was a highly significant difference in the PICF IL-1β levels between the 6th month and 12th month intervals (81.30 ± 47.08 pg/μl). This signifies that there was some form of subclinical inflammation around the implants which was detected by the biomarker. Hence, this suggests us that IL-1β can be a useful adjunctive diagnostic marker for assessing the early peri-implant health status.

However, further studies can be planned with larger sample size with longer follow-up period. Also, comparison between “healthy” and “peri-implantitis” groups would have helped us better correlate the IL-1β levels with clinical and radiographic parameters. Hence, the predictability of these results on the long term basis needs to be assessed by further research.

CONCLUSION

Within the limitations of the present research, assessment of the various parameters confirmed that all the implants had a healthy peri-implant status. Although the levels of IL-1β in PICF were elevated at the 12th month, they were well within the healthy range as observed by previous studies. This indicates that IL-1β, a biochemical marker, can be used as an adjunct to clinical and radiographic parameters in the assessment of EARLY inflammatory changes around implants.