Interleukins in Gingival Crevicular Fluid in Patients with Definitive Full-Coverage Restorations

Kai-Chiao J. Chang, DDS; Michelle A. Wheater, PhD; Levyee Cabanilla Jacobs, DDS; and Luis A. Litonjua, DDS

Many studies have investigated the effects of fixed partial dentures, specifically crown margin placement, in relation to the gingival margin and subsequent periodontal health. Crown margins can be placed supragingivally or subgingivally, and this placement may affect the ability of the patient to perform adequate oral hygiene. A retrospective study investigated the relationship of crowns or proximal restorations of at least 5 years on molar teeth and subsequent furcation involvement.¹ The authors found a higher prevalence of furcations and attachment loss on molars with restorations compared to molars without restorations. Schätzle et al conducted a 26-year longitudinal study of two cohorts consisting of good to moderate oral hygiene patients.² One cohort had subgingival restorations placed, while the other had supragingival restorations. The authors confirmed the long held concept that restorations placed below the gingival margin are detrimental to periodontal health. Although supragingival margins did not differ significantly compared to unprepared contralateral teeth, the subgingival crown margin resulted in greater bleeding and recession compared to control teeth.³

In a comparison of crown margins located at equigingival, supragingival, or subgingival levels, the subgingival margins appeared to have had the highest degree of periodontal pathogens in plaque, the greatest sulcus flow rate, the deepest probing depth, and higher levels of gingival inflammation.⁴ In comparison, supragingival margin placement had the least deleterious effects. Equigingival placement demonstrated some parameters similar to the subgingival margin group. In a long-term study of the oral hygiene, pocket depth, and attachment loss in patients treated with fixed dental prosthesis, 65% of crown margins were initially located subgingivally, compared to 41% located subgingivally 5 years later.⁵ The subgingival crowns had increased gingival index scores, pocket depth, and attachment loss compared to supragingival placement.

The inflammatory response of the gingiva may be observed by assessing changes in gingival crevicular fluid (GCF). The GCF acts as a defensive barrier in the gingival sulcus and protects oral tissues (including the junctional epithelium) against attack by bacterial invasion and trauma. GCF contains bacteria, desquamated epithelial cells, monocytes and other leukocytes, macrophages, and bacteria- and host-derived proteins.⁶ Cytokines, including interleukins, have been detected in GCF and identified as possible diagnostic markers of periodontal disease.^7-9

Interleukin (IL)-1 is a pro-inflammatory cytokine that serves a biological regulatory and inflammatory response, and exists in two forms: IL-1α and IL-1β.¹⁰ IL-1α is primarily produced by epithelial cells.¹¹ IL-8 is also a pro-inflammatory mediator that appears to be associated with periodontal destruction.¹² Multiple cell types synthesize and secrete IL-8 (eg, epithelial and endothelial cells, macrophages, fibroblasts, lymphocytes, and monocytes).^13,14 By analyzing IL-1β and matrix metalloproteinase (MMP)-2 levels, Moretti et al concluded that periodontal health is better in patients with supragingival restorations when compared with equigingival or subgingival restorations.¹⁵

There are relatively few published studies analyzing the interleukin content of GCF associated with the sites of crowns. The objective of this cross-sectional study was to determine and compare the levels of IL-1α and IL-8 in the GCF of patients who have full-coverage fixed restorations (crowns) with equigingival and supragingival margins in both periodontal health and disease.

Materials and Methods

The University of Detroit Mercy Institutional Review Board approved this research as expedited (IRB protocol approval #1112-49) before initiation of the study. The purpose and procedures of the study were explained to all potential study volunteers, and written informed consent was obtained from all of the study participants. This study included a total of 7 men and 5 women who were systemically healthy patients of the University of Detroit Mercy School of Dentistry from August 2010 to September 2011. Exclusion criteria included the use of antibiotics or the requirement for antibiotic prophylaxis and the use of nonsteroidal anti-inflammatory medication or contraceptives. Patients with a medical history of systemic inflammatory disease, diabetes, or other significant systemic disease, and patients who were pregnant, were also excluded.

As a component of the regular dental examination, clinical information relevant to this study was recorded, including periodontal status, smoking history, oral hygiene status, and plaque index. The patient’s periodontal and oral hygiene status was assessed on clinician determination. Plaque index (PI) was determined based on the method of O’Leary.¹⁶ Teeth from all quadrants of the mouth were measured, and 2 to 8 samples were collected per volunteer. In addition, probing depth in mm and the presence or absence of bleeding on probing (BOP) were determined for the study teeth. Probing depth measurements were obtained from mesio-buccal, mesio-lingual, disto-buccal, and disto-lingual sites using a conventional manual graduated periodontal probe directed parallel to the long axis of the tooth. Probing depths were rounded off to the nearest 1 mm. BOP was recorded as positive if bleeding was observed after 15 to 30 seconds of gentle probing. Clinical parameters were recorded and GCF samples were obtained by one examiner.

During their scheduled dental appointment, a total of 61 GCF samples were collected from the patients with definitive full-coverage restorations with two categories of crown margin placements. “Equigingival” margins defined a crown margin that was on the same level as the surrounding free gingiva, and “supragingival” margins defined a crown margin that was above the free gingiva. Of the 61 GCF samples that were collected, 19 samples were taken from the contralateral natural teeth without crowns to serve as the control. If these teeth were absent, the natural tooth adjacent to the contralateral tooth was used as a control. Twenty samples were collected from fixed full-coverage restorations with equigingival crown margins, and 22 samples were collected from fixed full-coverage restorations with supragingival crown margins. GCF samples were taken from distal, facial, mesial, and lingual sites.

The samples were collected before periodontal probing to prevent blood contamination. A tooth site selected for sampling was dried with a blast of air and isolated with cotton rolls. GCF samples were collected using specially designed PerioPaper strips (Oraflow Inc., www.oraflow.com). The strips were inserted into the gingival crevice until slight resistance was felt, and fluid was collected for 20 seconds. Care was taken to ensure the sterility of the PerioPaper strips when they were in contact with the patients. Any strips that were visually contaminated with blood were discarded. Because the amount of GCF varied, a chairside Periotron 8000 (Oraflow Inc.) was used to determine the volume of fluid taken up on each strip. The Periotron was calibrated, and arbitrary units were measured for each strip. The arbitrary units were converted to volume in μL using a standard curve according to the manufacturer’s instructions. Multiple sampling sites were obtained from patients from all quadrants, and each GCF sample was analyzed separately. The strips were stored in a microfuge tube at -20o C until analyzed.

Levels of IL-1α, IL-4, IL-6, and IL-8 in the GCF were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc., www.mdsystems.com). The PerioPaper strip samples were thawed, and 500-μl sample dilution buffer from the ELISA kits was added to the microfuge tubes. GCF was eluted from the strips by incubation at room temperature for 30 minutes. The strips were then subjected to centrifugation at 8,000 g for 10 minutes to pellet debris. Interleukin concentrations in duplicate GCF samples were determined from standard curves generated from the set of standards provided with each of the ELISA kits. IL-1α and IL-8 in all of the GCF sample concentrations (pg/mL) were within the detectable assay range of the kits. IL-4 and IL-6 concentrations were below the range of detection of the assay.

Analysis of variance (ANOVA) was used to compare interleukin concentrations between the control, supragingival, and equigingival GCF samples. Statistical analysis was completed using GraphPad Prizm software (GraphPad Software, Inc., www.graphpad.com) at a 5% level of confidence.

Results

Twelve adult patients were recruited for this pilot study. The volunteer’s ages ranged from 26 to 73 years, and included nonsmokers, former smokers, and current smokers. Demographic data for the study volunteers are summarized in Table 1. Probing depths ranged from 1 mm to 10 mm, and 40% of all of the sites exhibited BOP after the collection of GCF. The majority of GCF samples were taken from sites surrounding porcelain-fused-to-metal crowns (41 of 44 samples, 93.2%), with the remaining samples taken from sites surrounding gold crowns (3 of 44 samples, 6.8%).

All of the GCF samples tested were positive for IL-1α and IL-8 by ELISA and all samples as analyzed according to the kit protocols were within the sensitivity range of the assays. IL-1α and IL-8 concentrations were standardized across all of the samples for dilution of the test strip and were reported as pg/mL of the total sample to undertake statistical analysis of the data. Both interleukin concentrations showed wide biological variation. IL-1α concentrations ranged from 1.2 pg/mL to 474.3 pg/mL, while IL-8 concentrations ranged from 1.6 pg/mL to 347.8 pg/mL.

In comparing mean IL-1α concentrations taken from controls (60.4 ± 8.9 pg/mL), equigingival margins of crowns (110.5 ± 23.3 pg/mL), and supragingival margins of crowns (53.8 ± 9.7 pg/mL), the highest levels of interleukin were detected in GCF samples taken from equigingival crown margins (Figure 1). In GCF samples taken from gingival sulci with a probing depth of ≤ 3 mm, mean IL-1α concentrations were greater in equigingival margins (95.2 ± 17.0 pg/mL) compared to supragingival margins (45.7 ± 19.1 pg/mL) or controls (60.4 ± 8.9 pg/mL) (Figure 2). Similar observations were noted for GCF retrieved from sulci with a probing depth of > 4 mm. Mean IL-1α concentrations were greater in equigingival margins (129.2 ± 20.0 pg/mL) compared to supragingival margins (57.8 ± 11.3 pg/mL) or controls (59.0 ± 14.1 pg/mL) (Figure 3). For all analyses, mean IL-1α concentrations from supragingival margins were equivalent or less than control values, and concentrations from equigingival crown margins were greater than either control or supragingival values. However, statistical significance was not achieved, presumably because of the large standard deviation values.

In comparing mean IL-8 concentrations taken from controls (59.0 ±14.1 pg/mL), equigingival margins of crowns (131.4 ± 27.5 pg/mL), and supragingival margins of crowns (46.9 ± 9.7 pg/mL), the highest levels of interleukin were detected in GCF samples taken from equigingival crown margins (Figure 4). In GCF samples taken from gingival sulci with a probing depth of ≤ 3 mm, mean IL-8 concentrations were greater in equigingival margins (104.9 ± 37.5 pg/mL) compared to supragingival margins (37.4 ± 17.9 pg/mL) or controls (59.0 ± 14.1 pg/mL) (Figure 5). Similar observations were noted for GCF retrieved from sulci with a probing depth of > 4 mm. Mean IL-8 concentrations were greater in equigingival margins (154.9 ± 40.2 pg/mL) compared to supragingival margins (51.6 ± 11.7 pg/mL) or controls (59.0 ± 14.1 pg/mL) (Figure 6). For all analyses, IL-8 concentrations from supragingival margins were equivalent or less than control values, and concentrations from equigingival crown margins were greater than either the control or supragingival values. However, statistical significance was not achieved, presumably because of the large standard deviation values. IL-8 concentrations were higher than IL-1α concentrations in samples taken from crowns with equigingival margins.

Discussion

The development of periodontal disease is a multifactorial process that results in an inflammatory response.¹⁷ The primary etiology of periodontal disease is plaque. However, local contributing factors from dental restorations, including fixed prosthodontics with imperfect crown margins, margin placement, and crown contour, may further contribute to plaque retention.¹⁸ In addition to the presence or absence of restorations, crown margin placement has also been shown to significantly impact periodontal tissues. Schätzle et al confirmed that restorations placed below the gingival margin are detrimental to gingival and periodontal health.²

Although several studies have demonstrated the effects of margin placement on plaque accumulation, gingival conditions, probing depths, and attachment levels,^3-5,19 there are very few studies that have investigated the relationship of margin placement to the interleukin content of GCF. Moretti et al measured the levels of IL-1 and MMP-2 to evaluate the inflammatory response in sites where crowns were placed supragingivally, equigingivally, and subgingivally.¹⁵ They concluded that periodontal health is better in patients with supragingival restorations. Erdemir et al measured IL-6 and IL-8 concentrations in the GCF of teeth with fixed partial dentures and showed a reduction in IL-8—but not IL-6—after periodontal treatment.⁸ According to Dragoo and Williams, the large variation in both IL-1 and IL-8 concentration could be a result of local plaque retention factors from crown preparation, margin placement, and discrepancies.²⁰ In the current study, the goal was to investigate the effect of different levels of crown margin placement on interleukin levels in GCF.

The highest level of IL-1α was found in sites with equigingival margins of crowns, followed by the controls, and then the supragingival margins of crowns. Similar results were found for IL-8, where the equigingival margins of crowns presented with the highest inflammatory response, followed by controls, and then the supragingival margins of crowns. Since supragingival margins were associated with the lowest levels of interleukins in this study, crowns with any margins other than supragingival may be associated with greater periodontal changes based on the inflammatory response. However, the exact nature of this association is difficult to ascertain. It is possible that the increased interleukin levels seen in equigingival margins are a result of factors such as increased plaque retention. Using PI, the study patients’ oral hygiene was determined to be good (PI < 10%), fair (PI 11% to 50%), or poor (PI > 51%). Although Reitemeier et al suggested that oral hygiene before treatment, plaque formation, and margin placement affected the gingival health around posterior artificial crowns,¹⁹ this study suggests that PI and oral hygiene scores alone were not sole determinants for the observed interleukin concentrations. Because similar results were observed when comparing probing depths of < 3 mm and periodontal pockets with probing depths > 4 mm, this study suggests that probing depth may similarly not be the sole variable in disease progression.

This study demonstrated that GCF collected from sites of supragingival margins contained the lowest interleukin concentrations. This agrees with the study of Flores-de-Jacoby et al, which examined the effects of crown margin location on plaque.⁴ The authors reported that supragingival margins presented with the least deleterious effects. Additionally, Müller analyzed supragingival and equigingival crown placement on patients who had closely supervised periodontal maintenance therapy.²¹ Supragingival margins showed little or no signs of inflammation, and the microbial plaque observed was associated with healthy condition.

Conclusions

Statistically, no difference in IL-1α and IL-8 cytokine concentrations was observed in GCF associated with supragingival or equigingival crown margin placements. However, supragingival margins elicited less inflammatory response compared to controls or equigingival margins irrespective of oral health or probing depth. Crown margin placement selection may affect local plaque control and subsequently jeopardize the periodontal tissues supporting the tooth. Therefore, it is advised to consider margin selection as part of treatment planning to avoid localized periodontal destruction.

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About the Authors

Kai-Chiao J. Chang, DDS, MS
Adjunct Clinical Faculty
Department of Periodontics
Ostrow School of Dentistry
University of Southern California
Los Angeles, California
Former Graduate Student
Department of Periodontics and Dental Hygiene
School of Dentistry
University of Detroit Mercy
Detroit, Michigan

Michelle A. Wheater, PhD
Associate Professor
Department of Biomedical and Diagnostic Sciences
School of Dentistry
University of Detroit Mercy
Detroit, Michigan

Levyee Cabanilla Jacobs, DDS, MSD
Associate Professor
Department of Periodontics and Dental Hygiene
School of Dentistry
University of Detroit Mercy
Detroit, Michigan

Luis A. Litonjua, DDS
Adjunct Assistant Professor
Department of Periodontics and Dental Hygiene
School of Dentistry
University of Detroit Mercy
Detroit, Michigan