Proinflammatory cytokines (IL-1β and TNF-α) and chemokines (IL-8 and MIP-1α) as markers of peri-implant tissue condition

Abstract

Analysis of peri-implant crevicular fluid (PICF) offers a non-invasive means of studying the host response in peri-implant disease and may provide an early indication of patients at risk for active disease. This study examined the PICF levels of interleukin-1beta (IL-1β), tumour necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and macrophage inflammatory protein-1alpha (MIP-1α) in patients with non-manifesting inflammation, early and late stages of mucositis. The study group comprised 90 adult healthy volunteers with endosseal titanium implants inserted. Samples were taken from peri-implant sulcus using a filter paper technique. Implant tissues were categorized clinically as healthy, early mucositis or advanced mucositis. Clinical manifestations were determined by: gingival index and bleeding on probing, plaque index and radiographic analyses. Cytokine concentrations were assesed using commercially available enzyme-linked immunosorbent assay kits. Patients from the control group (healthy patients) have significantly lower concentrations of IL-1β, TNF-α, IL-8 and MIP-1α in PICF compared with both groups with mucositis. Positive correlation was noted in the control group between IL-1β and TNF-α and between MIP-1α and IL-8 in the group with early mucositis. The results suggest that cytokines could be prognostic markers of implant failure.

Current models of peri-implant pathogenesis use modern concepts of immunobiology to explain the processes and consequences of disease progression. Investigators are searching for valid biological diagnostic markers of high sensitivity that can indicate the presence of the disease process before extensive clinical damage occurs.

The marginal inflammatory tissue reactions in implantology are similar to those encountered in gingivitis and periodontitis. Plaque formation patterns identified on implants are similar to those observed on teeth . As soon as plaque accumulation begins, neutrophils are recruited to the peri-implant pocket or the gingival crevice because of the chemotactic peptides released by the bacteria. As bacteria damage the epithelial cells, they cause the release of cytokines that further attract leucocytes (predominantly neutrophils) to the crevice. They can phagocytose and digest bacteria and therefore, remove these bacteria from the pocket. If the neutrophil becomes overloaded with bacteria, it degranulates thus causing tissue damage because toxic enzymes are released from the neutrophils.

If there is an overload of microbial plaque, the neutrophils and the barrier of epithelial cells will not be sufficient to control the infection. In such instances, the peri-implant tissues will become inflammed and this is clinically diagnosed as peri-implant mucositis. Spreading the inflammation from the marginal gingiva into the supporting peri-implant tissues results in bone destruction and loss of bone attachment; a process termed peri-implantitis . Peri-implantitis has also been described as a site-specific infection presenting many features in common with chronic adult periodontitis, but the soft tissue inflammatory lesion and the bone loss is larger around implants. Positive correlation has been found between plaque accumulation and marginal bone loss around dental implants.

Peri-implantitis is an inflammatory reaction affecting the tissues surrounding dental implants. Whilst peri-implant mucositis is a reversible inflammation of the soft tissues surrounding the dental implant, peri-implantitis is an inflammatory reaction affecting the tissues surrounding osseointegrated dental implants resulting in loss of supporting bone . Early and late mucositis are characterized by the change in composition of the microbial plaque, with increased Gram-negative microorganisms. These Gram-negative bacteria activate a localized host response. The products of inflammatory responses occurring within the periodontium during disease are also present in peri-implant crevicular fluid (PICF) in the case of mucositis. Analysis of inflammatory mediators in PICF has also been used to compare peri-implant tissue health and disease .

Amongst many inflammatory and immune mediators identified in PICF, cytokines have attracted particular attention. Cytokines are proteins secreted by the cells of innate and adaptive immunity that mediate many of the functions of these cells. They are intercellular messengers that are essential for the pathogenesis of many diseases including peri-implantitis. These soluble mediators of immune reactions present in PICF are produced as a result of the physiological interaction of gingival epithelium and local leukocytes with dental plaque and oral microorganisms. They also play important roles in tissue homeostasis. In periodontology and implantology they are involved in inflammation-related alteration and repair of periodontal or peri-implant tissues. Certain cytokines have been proposed as potentially valid diagnostic or prognostic markers of periodontal or peri-implant tissue destruction .

Owing to the site-specific nature of the peri-implant disease process, this network must be controlled by local processes. Analysis of gingival crevicular fluid (GCF) or PICF offers a non-invasive means of studying the host response in periodontal and peri-implant disease and may provide an early indication of the patient at risk for active disease. Peri-implant sulcus fluid analysis may help in detecting early metabolic and biochemical changes that are not studied sufficiently. Different biochemical markers in GCF and PICF have been widely investigated .

Most of the studies have investigated the role of cytokines in GCF in periodontal disease, fewer have studied cytokines in PICF in peri-implantitis. Local host response in peri-implant bacterial infection is a relatively new area of research.

Interleukin-1beta (IL-1β) and tumour necrosis factor alpha (TNF-α) are proinflammatory cytokines that appear to have a central role in periodontal tissue destruction .

The biological effects of IL-1β depend on its tissue concentration. The properties of these cytokines that relate to tissue destruction involve stimulation of bone resorption and induction of tissue-degrading proteinases . TNF-α is a proinflammatory cytokine with similar properties to IL-1β. It is the main mediator in response to Gram-negative bacteria and concentration of TNF-α reflects the amount of bacteria and the stage of inflammation.

During the past decade, a superfamily of leukocyte chemotactic proteins, known as chemokines has been identified. Chemokines selectively attract and activate different leukocyte subpopulations and are key mediators of a variety of patho-physiological conditions, including inflammation .

Interleukin-8 (IL-8), a member of the neutrophil-specific CXC subfamily of chemokines, is a potent neutrophil chemotactic and activating factor that has a crucial role in the selective recruitment and activation of neutrophils and in routing them to the gingival sulcus. Macrophage inflammatory protein (MIP-1α) is a monocyte chemoattractant, a member of the β or CC subfamily of chemokines that act as potent progenitor cells. These chemokines cause the selective migration of human monocytes and lymphocytes towards sites of inflammation and are the mediators of various patho-physiological conditions.

The aims of the present study were to determine and compare the levels of IL-8, MIP-1(, IL-1β and TNF-α in PICF samples from healthy patients, patients with early and patients with late stage mucositis and to determine any correlation between the levels of IL-1β, TNF-α, MIP-1α and IL-8 in different stages of peri-implant tissue inflammation.

Materials and methods

Patient selection

The study population included 90 adult healthy volunteers (nine females and 81 males), mean age 55 years, who had had at least one endosseal titanium implant with a purity level of 2/ASTM (American Society for Testing and Materials) (99.98%) and a sand-blasted, large-grit, acid (SLA) etched surface inserted. Implants were 4.5 mm in diameter, 13.5 mm long with 4 threads and were inserted into bone type II. During the study period the patients were instructed to maintain oral hygiene properly.

Patient selection was based on clinical signs of inflammation. They were selected from the regular check-up schedule for patients treated with osseointegrated dental implants. All participants were systemically healthy and had not received antibiotics during the previous 6 months. After thorough instruction about the purpose and protocol of the study, participants were asked to give their consent in written form. The study protocol was approved by the Ethics Committee in MMA, Belgrade.

In all patients, evaluations were made after delivering the suprastructures, which were functional for 12–36 months. Evaluations included assessment of gingival index (GI) and bleeding on probing (BOP), plaque index (PI) and radiographic analyses. Clinical measurements of GI, PI and BOP were taken at four sites (mesial, buccal, distal and lingual). Periapical X-rays were analysed and revealed no bone loss in any patient. Gingival inflammation was scored following crevicular fluid collection using the criteria for the GI system . In this system each site is given a score from 0 to 3, as follows: GI 0 is normal gingiva; GI 1 is mild inflammation, a slight change in colour, slight oedema, but no bleeding on probing; GI 2 is moderate inflammation, redness, oedema and glazing, and bleeding on probing; GI 3 is severe inflammation with marked redness and oedema, ulceration and a tendency for spontaneus bleeding.

The patients were classified into three groups: control group I (49 participants) included patients with clinically healthy gingival tissue around dental implants (GI = 0); group II (30 participants) included patients with early stage mucositis (GI = 1); and group III (11 participants) included patients with advanced stage mucositis (GI = 2).

GI 3 was not studied because it is of limited value to peri-implantitis. BOP was measured dichotomously within 30 s after probing. Prior to crevicular fluid collection, supragingival plaque was scored using the PI where: 0 is no plaque in the gingival area; 1 is a film of plaque adhering to the free gingival margin and adjacent area of the tooth (implant); 2 is moderate accumulation of soft deposits within the gingival pocket, on the gingival margin and/or adjacent tooth (implant crown) surface, which can be seen by the naked eye; and 3 is abundance of soft matter within the gingival pocket and/or on the gingival margin and adjacent tooth surface. PI usually correlates with GI, but does not accord with gingival status in every case.

The PI values in group I were 0 (40 patients) and 1 (nine patients). In group II the values were 0 (three patients), 1 (23 patients) and 2 (four patients) and in group III they were 1 (four patients) and 2 (seven patients) ( Table 1 ). All clinical data were recorded by one examiner.

Table 1
PI and PD values in different stages of peri-implant tissues condition .
PI = 0 PI = 1 PI = 2 PD ≤ 3 PD > 3 < 4 PD ≥ 4 Σ
Control group (group I) GI = 0 40 9 0 49 0 0 49
Early peri-implantitis (group II) GI = 1 3 23 4 0 30 0 30
Advanced peri-implantitis (group III): GI = 2 0 4 7 0 0 11 11

PICF sampling

The PICF samples were obtained from the patients with healthy gingiva and from patients in different stages of tissue inflammation. Sampling was carried out after delivering the suprastructure. PICF was sampled using a filter paper technique. PICF samples were taken from peri-implant sulci. The gingiva were dried by air and cotton pellets for 1 min before sampling and the area isolated using cotton rolls. A paper strip of standard length and height (Periopaper, Pro Flow, Amityville, NY, USA) was inserted into the peri-implant sulcus until mild resistance was felt and left in place for 30 s. Strips macroscopically contaminated with blood or saliva were discarded. Strips were measured for fluid volume with calibrated Periotron 6000 (Interstate Drug Exchange, Amityville, NY, USA). In patients with healthy gingiva, the quantity of gingival fluid was lower than in those with gingival inflammation. The sample strip was inserted into plastic sealable Eppendorf tubes and eluated in 100 μl of sterile NaCl. Following 10 s of vortexing, eluates were centrifuged and the strips were removed. The samples were stored at −20 °C.

Enzyme-linked immunosorbent assay (ELISA)

Proinflammatory cytokine (IL-1β and TNF-α) and chemokine (IL-8 and MIP-1α) concentrations in PICF eluates were assessed using commercially available ELISA kits (R&D, SAD) Quantokine Immunoasay for Human IL-1 beta (cat. number DLB50), Human TNF-alpha (cat. number DTA50), Human IL-8 (cat. number D800C), Human MIP-1 alpha (cat. number DMA00). They detect levels of cytokines as low as 0.3 pg/ml using the quantitive sandwich enzyme immunoassay technique. Monoclonal antibodies specific for those cytokines and chemokines being studied were pre-coated into a microplate. Standards and samples were pipetted into the wells and incubated for 3 h at room temperature. Any IL-1β, TNF-α, IL-8 or MIP-1α were bound by the immobilized antibodies.

After washing off any unbound substances, an enzyme-linked polyclonal antibody specific for that cytokine or chemokine was added to each well. The plate was incubated at room temperature for 2 h and the wells re-washed. The substrate solution was added to the wells and the colour developed was proportional to the amount of IL-1β, TNF-α, IL-8 or MIP-1α bound in the initial step. After 20 or 30 min incubation, stop solution (sulfuric acid) was added and the reaction arrested. The intensity of the colour was measured using spectrophotometry (450/650 nm, ELISA processor II, Boehring, Germany).

Statistical analyses

Statistical analyses were performed using a commercial statistics package for PC computers (Stat for Windows, R4.5, SAD). Differences in cytokine levels between diseased and non-diseased sites (samples and controls) were evaluated by Mann–Whitney, Wilcoxon rank sum W test. The Pearson correlation coefficient was used to study the correlations between IL-1β and TNF-α levels and clinical parameters.

Concentrations of chemokines were calculated in relation to the volume of PICF. The values given for each chemokine, represent the median and range values.

Results

Concentrations according to the stage of mucositis

Concentrations of cytokines were calculated in relation to the PICF volume. Patients from the control group had significantly lower concentrations of IL-1β, TNF-α, MIP-1α and IL-8 in PICF compared with both groups with mucositis ( p < 0.01 for IL-1β and p < 0.001 for TNF-α, MIP-1α and IL-8). Mean PICF values were significantly higher in the group with advanced mucositis compared with the group with early mucositis ( Tables 2 and 3 ).

Table 2
Concentrations of proinflammatory cytokines (IL-1β and TNF-α) in PICF of patients with different stages of peri-implant tissue condition.
Patients X SD med SE N
IL-1β (pg/ml)
Control 12.6 14.8 7.0 2.1 49
Early peri-implantitis 178.7** 208.1 109.0 38.6 30
Advanced peri-implantitis 1705.3 ♦♦, ** 2410.6 750.0 726.8 11
TNFα (pg/ml)
Control 0.8 1.85 0.0 0.3 49
Early peri-implantitis 19.8*** 23.6 12.0 4.4 30
Advanced peri-implantitis 130.4 ♦♦♦, *** 74.1 132.0 22.3 11
** p < 0.01 compared with control group.
♦♦ p < 0.01 compared with early peri-implantitis.
*** p < 0.001 compared with control group.
♦♦♦ p < 0.001 compared with early peri-implantitis.
X, mean; SD, standard deviation; med, median; SE, standard error.

Table 3
Concentrations of chemokines (MIP1-α and IL-8) in PICF of patients with different stages of peri-implant tissue condition.
Patients X SD med SE N
MIP1-α (pg/ml)
Control 8.2 8.3 5.0 1.2 49
Early peri-implantitis 61.8*** 28.3 58.0 5.3 30
Advanced peri-implantitis 150.5♦♦♦,*** 50.1 155.0 15.1 11
IL8 (pg/ml)
Control 47.5 29.3 42.0 4.1 49
Early peri-implantitis 208.8*** 182.5 161.0 33.9 30
Advanced peri-implantitis 1659.0♦♦♦,*** 1506.8 1299.0 454.3 11
*** p < 0.001 compared with control group.
♦♦♦ p < 0.001 compared with early peri-implantitis.
X, mean; SD, standard deviation; med, median; SE, standard error.

Concentrations according to PI values

The patients with a PI value of 0 had significantly lower concentrations of IL-1β, TNF-α, MIP-1α and IL-8 in PICF compared with those patients whose PI was 1 or 2. Mean concentrations of IL-1β, TNF-α, MIP-1α and IL-8 in PICF were statistically significantly higher in group III ( p < 0.01) compared with patients in group II ( p < 0.001) ( Tables 4 and 5 ).

Table 4
Concentrations of proinflammatory cytokines (IL-1β and TNF-α) in PICF of patients with different PI values.
Patients X SD med SE N
IL-1β (pg/ml)
PI 0 14.0 19.1 6.5 2.9 43
PI 1 156.8** 207.1 84.5 35.5 36
PI 2 1705.3 ♦♦, ** 2410.6 750.0 726.8 11
TNF-α (pg/ml)
PI 0 1.0 2.5 0.0 0.4 43
PI 1 24.6*** 43.9 5.5 7.5 36
PI 2 100.0 ♦♦♦, *** 77.0 96.5 27.2 11
** p < 0.01 compared with group with PI 0.
♦♦ p < 0.01 compared with group with PI 1.
*** p < 0.001 compared with group with PI 0.
♦♦♦ p < 0.001 compared with group with PI 1.
X, mean; SD, standard deviation; med, median; SE, standard error.

Table 5
Concentrations of chemokines (MIP-1α and IL-8) in PICF of patients with different PI values.
Patients X SD med SE N
MIP1-α (pg/ml)
PI 0 8.2 8.3 5.0 1.2 43
PI 1 61.8*** 28.3 58.0 5.3 36
PI 2 150.5 ♦♦♦, *** 50.1 155.0 15.1 11
IL8 (pg/ml)
PI 0 50.7 44.5 40.0 6.7 43
PI 1 215.0*** 206.7 123.5 35.5 36
PI 2 1441.1 ♦♦, *** 1574.4 1028.5 454.5 11
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Feb 8, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Proinflammatory cytokines (IL-1β and TNF-α) and chemokines (IL-8 and MIP-1α) as markers of peri-implant tissue condition
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