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Year : 2021  |  Volume : 24  |  Issue : 8  |  Page : 1181-1187

Expression Level and Clinical Significance of Inflammatory Cytokines and Biochemical Markers in Gingival Crevicular Fluid During Different Crown Adhesion Patterns of Dental Implant

Department of Stomatology, The Third Hospital of Hebei Medical University, Hebei, China

Date of Submission01-Apr-2020
Date of Acceptance17-Jun-2020
Date of Web Publication14-Aug-2021

Correspondence Address:
Dr. X Hou
Department of Stomatology, The Third Hospital of Hebei Medical University, 139 Ziqiang Road, Shijiazhuang, Hebei - 050051
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njcp.njcp_152_20

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Objective: The aim of this study was to investigate the expression level and clinical significance of inflammatory factors and biochemical markers in gingival crevicular fluid during different crown-binding styles in dental implant patients. Methods: A total of 38 patients with posterior tooth loss and implant repair were recruited and divided into two groups according to the different ways of crown bonding, including 19 prostheses (19 patients) in the adhesive retainer group and 19 prostheses (19 patients) in the modified adhesive retainer group. Moreover, the peri-implant gingival sulcus fluids of each group of patients were collected at 7, 15, 30, 60, and 90 d of post-treatment, and the expression level of each cytokine as well as biochemical marker were analyzed by enzyme-linked adsorption method, respectively. Results: Compared with the control group, the peri-implant plaque index and gingival bleeding index were decreased in the observation group. In addition, the secretion of peri-implant gingival crevicular fluid in the observation group was significantly higher than that of the control group. The level of IL-6, TNF-α expressions in peri-implant gingival crevicular fluid were gradually decreased with follow-up time, and the rate of decline gets slow at 15 h after operation. The TGFα in peri-implant gingival crevicular fluid in the two groups began to increase at 7 d, reached a peak at about 15 d, then slowly decreased and stabilized after 60 d. While the OCN was gradually increased during the whole detection process, slowly released before 30 d, then increasingly released and maintained at a peak state after 60 d. All the above differences were statistically significant (P < 0.05). Conclusion: Different crown-binding patterns of implant teeth have a significant effect on the secretion amount of peri-implant gingival crevicular fluid and the expression level of inflammatory cytokines as well as biochemical markers.

Keywords: IL-6, OCN, peri-implant gingival crevicular fluid, TGFα, TNF-α

How to cite this article:
Wang L, Fan S, Yang J, Liu Q, Wang F, Hou X. Expression Level and Clinical Significance of Inflammatory Cytokines and Biochemical Markers in Gingival Crevicular Fluid During Different Crown Adhesion Patterns of Dental Implant. Niger J Clin Pract 2021;24:1181-7

How to cite this URL:
Wang L, Fan S, Yang J, Liu Q, Wang F, Hou X. Expression Level and Clinical Significance of Inflammatory Cytokines and Biochemical Markers in Gingival Crevicular Fluid During Different Crown Adhesion Patterns of Dental Implant. Niger J Clin Pract [serial online] 2021 [cited 2022 Aug 15];24:1181-7. Available from:

In recent years, dental implant technology has been widely applied in clinic, and the success rate of implant treatment in 10 years is about 89%–95%.[1] To further improve the life cycle of dental implants, it is very important to analyze and find out the impact factors which affect the life cycle of implant tooth. It is well known that at present, there are two kinds of bonding methods for implant crown restoration, that is, bonding retention and modified bonding retention. The two bonding methods have their own advantages and disadvantages, and the adhesive retention is better than the modified adhesive retention. The former may have a greater risk of peri-implant disease due to residual overflow adhesive. It has been noted that the changes of cytokines were detected in gingival crevicular fluid caused by the alteration of periodontal tissue and the dynamics between osteogenesis and osteoclast caused by mechanical force during initial loading. According to previous study,[2] the levels of inflammatory factors interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF-α) in gingival crevicular fluid have been dynamically altered, and these bioactive factors have been involved in tooth movement and alveolar bone reconstruction in the early days.[2] Transforming growth factor α (TGF α) is one of the key factors to promote the initial mechanism to bone transformation, while osteocalcin (OCN) is responsible for the binding of calcium ions to the initial matrix, which acts as an osteogenic marker and is released early in loading and is important for matrix mineralization.[3] Early stage of inflammation is necessary for bone regeneration, while chronic inflammation can cause periodontal and peri-implant tissue damage, which is not conducive to the prolongation of implant life cycle.[4] Therefore, the purpose of this study is to investigate the effect of different crown adhesive patterns on the expression levels of inflammatory factors (IL-6, TNF-α) as well as biochemical markers (TGFα, OCN) in gingival crevicular fluid and their clinical significance.

   Subjects and Methods Top

Research object

38 patients were recruited with posterior tooth loss who have been performed dental implant pending single crown repair in the stomatology department of the third hospital of Hebei Medical University from December 2016 to November 2017. The program has been approved by the hospital ethics committee (No. 2018-021-1). All patients volunteered to participate in the trial, understood the method and purpose of this study, signed informed consent and cooperated actively. The patients were divided into two groups according to the bonding mode of the crown. Control group: 19 prostheses (19 patients, 12 males, 7 females, mean age 44.2 ± 8.3); observation group: 19 prostheses (19 patients, 13 males, 6 females, mean age 43.7 ± 8.2).

Entry criteria

Inclusion criteria :1) good health, no history of radiotherapy for head and neck, no systemic disease; 2) good occlusal relationship, normal space of missing teeth, no elongation of occlusal teeth; 3) preoperative assessment of dental plaque and gingival index were 0 or 1; no antibiotics were used after implant completion, no periodontal treatment; 4) no smoking habits or less than 10 cigarettes per day; 5) good patient compliance, healthy living habits; 6) women were not in pregnancy; 7) there were contralateral teeth in position.

Treatment methods

Treatment methods: Both groups of patients were planted with ITI full thread columnar teeth, the implant transfer impression was taken by non-window type impression method, the healing base was unloaded, the suitable mold removal rod was selected to install on the implant and tighten the screws, the cotton ball was used for temporary sealing, the impression was taken by DMG silicone rubber and sent to the processing plant for production. The bonding surface of the bonding retainer group is complete without leaving a central screw hole, and the crown is directly bonded to the base table in the mouth. In the modified bonding group, the crown was first bonded to the abutment outside the mouth and then fixed to the implant through the central screw hole retained on the crown.

Sample collection

Cut Whatman 3 filter paper into strips 2 mm × 15 mm and place in a clean container. Gingival sulcus fluid was collected by the same dentist after training. The samples were collected in the morning (The patients were required to suspend oral hygiene maintenance measures such as brushing their teeth on the day of collection), the plaque was scraped off the gingival margin area of the prepared tooth, and the specimens were collected by immersing a spare filter paper in subgingival sulcus near the buccal side about 40 s. The samples were quickly put into EP sterilized tube (Blood stains and saliva contaminated samples should be excluded when samples were collected), marked and put into the -80 °C refrigerator for further use.

Determination of IL-6, TNF-α, TGFα and OCN mass concentrations in gingival crevicular fluid

The IL-6, TNF-α, TGFα and OCN concentrations in the samples were detected by ELISA double anti sandwich method. 1. Add the sample to be tested, the negative control and the standard (positive control) to the 96 well plate with shaking for 120 min. 2. Then add the primary antibody and shake for 60 min. 3. Add enzyme standard working fluid and shake for 60 min. 4. Add substrate 100 μL in each well, avoid light 10 min, and then add termination solution. 5. Reading meter measurement optical density value at 450 nm wavelength, drawing the standard curve to calculate the IL-6, TNF-α, TGFα and OCN concentrations in the sample as the baseline value for statistical analysis.

Other observation indicators

Weight of gingival crevicular fluid: Cut Whatman1 filter paper into 2 mm × 8 mm filter strip, which were irradiated by UV light for 2 hours and then packed in the aseptic operating table. Each tube was put in four filter strips, numbered and weighted (recorded as m1) with electronic analysis balance (accurate to 0.01 mg) for use. All patients gargle with clear water and scrape off the stones as well as plaque attached to the neck of the sampled teeth. The cotton ball is dampened, and then gently blowing dry on the cheek and neck of the tooth to be tested. Inserting the front end of 2 × 8 mm sterile filter strip into between the sample implant and the gingival sulcus of natural tooth proximal buccal point with a force of about 20 g until encountering slight resistance. After 40 s, the filter strip was taken out and quickly put into the original sterilized EP tube. Repeat the procedure with a new filter strip after 1 min interval, collect the gingival crevicular fluid several times (discarding samples contaminated with the blood and saliva) and put it in the original EP tube, weigh it immediately after collection (recorded as m2). Calculate the weight of gingival crevicular fluid (m2-m1).

Plaque index (PLI), (Each implant was examined on gingival at four sites, i.e., buccal lingual side and near distal gingival margin area. Each group of tested implants was examined at 76 sites and recorded): 0) No plaque; 1) Thin plaque is on the tooth surface of the margin area, but not visible, if the plaque can be scraped on the side of the probe tip; 2) Moderate plaque on the edge or adjacent surface; 3) Large number of plaque and soft scale on the inner or gingival margin area and adjacent surface.

Sulcus bleeding index (SBI), each tooth was examined at four sites of the gingiva, namely the buccal lingual side and the proximal distal gingival margin area. 76 sites in each group of tested implants and natural teeth were examined and recorded. If the locus of SBI is more than or equal to 2, there will be gingivitis. The score is as follows: 0 = Margin and gingival papilla have healthy appearance, no bleeding after probing gingival sulcus; 1 = Margin and gingival papilla show mild inflammation, no bleeding after probing gingival sulcus; 2 = Gingiva show mild inflammation, no swelling or edema, punctate bleeding after probing; 3 = Gingiva show moderate inflammation, color change and mild edema, bleeding after probing, bleeding within gingival sulcus; 4 = Severe inflammation of the gingiva, there are not only the color changes, but also obvious swelling, bleeding after exploration, and overflowing the gingival sulcus; 5 = Gingival colored changes, marked swelling, sometimes ulcers, bleeding or automatic bleeding after probing.

Statistical analysis

The data collected in this study were statistically analyzed by using SPSS 20.0 software package, in which the measurement data were expressed as mean ± standard deviation (X ± S), and the comparison between the two groups was tested by two independent samples t. Counting data were recorded in the form of rate, and the comparison between the two groups was tested by Fisher exact probability method. A– value of P < 0.05 was statistically significant.

   Results Top

General comparison of information

A total of 38 patients were included in this study, 19 in the control group and 19 in the observation group, and there was no significant difference in general data such as sex and age between the two groups (P > 0.05). It was indicated that the two sets of information are comparable. [Table 1]
Table 1: Comparison of general data of two groups

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Differences in peri-implant plaque indices between the two groups

Compared with the control group, the peri-implant plaque index and gingival bleeding index were decreased in the observation group, and the difference was statistically significant (P < 0.05) [Table 2] and [Figure 1].
Figure 1: Differences between peri-implant plaque index and gingival bleeding index under two different bonding modes (*P < 0.05)

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Table 2: Differences between peri-implant plaque index and gingival bleeding index under two different bonding modes

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The difference of gingival crevicular fluid weight and IL-6, TNF-α, TGFα and OCN expression between the two groups

There was no significant difference in the amount of gingival crevicular fluid secretion and IL-6, TNF-α, TGFα as well as OCN expression in gingival crevicular fluid between the two groups at 7 d (P > 0.05). The level of IL-6, TNF-α expression in peri-implant gingival crevicular fluid were gradually decreased with follow-up time, the rate of decline gets slow at 15 h after operation, and the difference between the two groups was statistically significant (P < 0.05). The TGFα in peri-implant gingival crevicular fluid in the two groups began to increase at 7 d, reached a peak at about 15 d, then slowly decreased and stabilized after 60 d, and the difference between groups was statistically significant (P < 0.05). While the OCN was gradually increased during the whole detection process, slowly released before 30 d, then increasingly released, and maintained a peak state after 60 d. The difference was statistically significant (P < 0.05). The weight of two groups of gingival crevicular fluid and the content of TNF-α, IL-6, TGF α and OCN in the gingival crevicular fluid around the implant are shown in [Table 3] and [Figure 2].
Figure 2: Differences in the weight of gingival crevicular fluid and the expression of IL-6, TNF-α, TGFα and OCN between the two groups (*P < 0.05)

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Table 3: The difference of gingival fluid weight and IL-6, TNF-α, TGF α and OCN expression between two groups

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   Discussion Top

Effect of crown bonding on peri-implant tissue health

At present, the common methods of implant restoration are adhesive fixation, screw retention and modified adhesive retention. The classic central screw retainer belongs to a section structure, and its retainer mode is directly connected to the superstructure through the central screw hole by a suitable retainer screw. The central screw retaining crown bridge prosthesis can be removed and cleaned by the dentist at any time. If there is lesion to be found clinically, the crown can be removed and treated immediately. Some scholars[5] have studied that the edge bone of the adhesive retainer lost less than that of the screw retainer, and long-term clinical observation found that the retainer of the adhesive retainer was higher. Also, some other scholars have found that there is no significant difference in bone resorption around the peri-implant under the two conditions, but it has been abandoned due to the limited indication of central screw fixation.[6] In recent years, it has been found that it is difficult to completely remove the adhesive which overflows in the gingival crevicular when the adhesive is fixed, which brings trouble to the clinical work.[7]

Because of the current pursuit in aesthetics, clinicians usually place the crown edge of the implant at 1-2 mm below the gingival, but some scholars[8] stated that for the bonded retainer, the experienced doctors can not completely remove the crown when the crown edge is located at 1.5-3 mm below the gingiva, and there are many scratches left on the abutment during the cleaning process, which may cause plaque accumulation and affect the health of the surrounding tissue of the implant.[9] Therefore, in order to avoid the adhesive residue and facilitate the later maintenance of the implant, another modified adhesive retention method has been proposed. Compared with the adhesive retention method, there is no residual adhesive in the gingival sulcus, and the abutment and crown can be easily removed from the implant by removing the central screw for easy repair and maintenance.[10] The analysis of peri-implant plaque index and gingival bleeding index in different crown-bonded dental implants showed that the peri-implant plaque index and gingival bleeding index in the observation group were less, compared with the control group (P < 0.05). This finding is also consistent with the result of previous findings, suggesting that the improved bonding retention is more conducive to the health of the implant and its surrounding tissues.

Changes of cytokines and biochemical markers in peri-implant gingival crevicular fluid

The level of IL-6 reached a peak in d 7 after operation, then decreased slowly. The rate of change slowed down after 15 d, and remained stable after 60 d. Firstly, the increase of postoperative IL-6 may be attributed to the biochemical response of mechanical stress to kinds of cells, since IL-6 induces the release of histamine by mast cells at the inflammatory site, and then triggers early vasodilation and increased vascular permeability.[11] Studies have shown that the interleukin IL-6 in patients with peri-implant inflammation have higher levels than that of healthy individuals, because IL-6 is an inflammatory factor that can cause inflammation around the implant. There are significant increases once inflammation occurs in the peri-implant tissue. And so IL-6 levels are associated with the severity of inflammation of tissues around the implant.[12] Previous research[13] suggested that the synthesis of proinflammatory cytokine IL-6 was observed during orthodontic exertion, leading to further bone resorption. The related molecular mechanism of its existence may be the fact that IL-6 up-regulates the levels of nuclear factor KB ligands (RANKL receptor activators), which will directly cause the absorption of surrounding bone. The content of the IL-6 has been reduced to half of the original at day 15, and after that the change of the measured concentration is small. This may be due to the complex effects of cytokines on various target cells that exhibits simultaneous negative as well as positive bidirectional regulation, and IL-6 receptor antagonists (IL-1Ra) inhibit IL-6 activity by binding to the IL-6 receptor (IL-1R).[14] This dual effects of positive or negative regulation may regulate the process of remodeling bone after synergistic or additive action, and the resulting feedback mechanism may prevent the excessive increase of inflammatory mediators.[15] A significant increase in the IL-6 level during implant implantation and subsequent decreasing trend suggest the adaptability of peri-implant tissues to stimulation.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine produced by activated monocytes, macrophages, and osteoblasts. In addition to bone resorption, TNF-α also stimulates fibroblasts to produce collagenase, which plays an important role in regulating and enhancing the inflammatory response of periodontal and peri-implant tissues. It has been reported that any changes of gene modification produced by TNF-α after infectious stimulation, can significantly influence the extent of inflammatory response and clinical outcomes.[16] In our experiment, the level of TNF-α was increased significantly in the early loading period, indicating that the implant created immune stimulation to the body, leading to chemotaxis of inflammatory factors and enhancing the inflammatory response of surrounding tissues. Some studies have shown that TNF-α can activate F-B signaling pathways, promote the differentiation of bone marrow mesenchymal cells into osteoclasts, and stimulate endothelial cell secretion PGE2, further aggravating bone absorption.[17] The level of TNF-α was decreased at day 15 and maintained at a certain level after 60 d, consistent with the change trend of IL-6. It has been previously reported that IL-6 could stimulate monocytes/macrophages to produce more TNF-α, as well as increase IL-6 expression, which is similar to our findings.[14]

TGFα has reached the maximum value at day 15 after detection, probably because in the first stage of bone binding, the initial matrix is composed of fibroblast-like cells, which provides template TGFα for future bone mineralization.[18] To control the factors involved in the initial matrix-to-bone transformation, some scholars have shown that it directly affects the production of RUNX2 gene transcription factors in chondrocytes, thus mediating the release of matrix metalloproteinases (MMP-9,13 and 14) and stimulating angiogenesis.[19] Furthermore, the TGF family is associated with osteoblast proliferation, differentiation, activation, and collagen synthesis, which is also consistent with our results. After the peak, the TGFα level was decreased significantly and stabilized after 60 days. This result suggests that immediate loading stimulates the first stage of ossification, and TGFα regulation allows it to move rapidly into the next stage.

Another important early cytokine is OCN, which is involved in the further mineralization of calcium ions binding into the matrix. Previous post-implant studies in dogs have found the higher levels of OCN mRNA in peri-implant bone tissue, calling OCN as a mechanical response gene, which increases OCN concentrations when subjected to mechanical stimulation.[20] OCN concentrations in this study were increased after initial loading and gradually increased, confirming the formulation of mechanical responsive genes.

The difference between SBI and PLI in two different bonding modes was statistically significant, and the amount of IL-6, TNF-α as well as gingival crevicular fluid showed differences, which indicated that the level of IL-6 as well as TNF-α in peri-implant and the amount of gingival crevicular fluid could be used as the detection index for early peri-implant stimulated by susceptibility factors, which was more sensitive than the clinical index. To some extent, it might reflect the healthy state of peri-implant tissue. Importantly, it provides evidences for future clinical diagnosis and treatment that we can detect IL-6 and TNF-α levels in high-risk post-implant repair patients, intervene if necessary, interrupt or slow down the occurrence and development of peri-implant diseases, so as to avoid serious bone resorption or damage caused by implant repair failure. It also can be applied as a tracking index for case observation to determine the long-term periodontal health status.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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