Possible role of gingival crevicular fluid levels of Chemerin and Fibroblast growth factor 21 as biomarkers of periodontal disease in diabetic and non-diabetic patients. A diagnostic accuracy study

Introduction:

Periodontitis is an extensive damaging inflammatory condition of the anatomy surrounding and supporting the teeth, that is initiated due to the interruption of normal homeostasis by abundant bacterial classes existing in subgingival dental plaque (Darveau, 2010), and is altered by genetic and environmental factors(Darveau, 2010, Pihlstrom et al., 2005). This leads to tissue injury involving loss of connective tissue attachments and alveolar bone damage, frequently resulting in compromised mastication and ultimately tooth loss (Pihlstrom et al., 2005)

Periodontitis is characterized by a variety of cytokines, chemokines, arachidonic acid metabolites, and inflammatory mediators including proteolytic enzymes which are released as a response to the activities of the tissue defense systems against the microbiological agents. These released molecules were blamed for soft tissue degradation and bone resorption in the host tissue  (Lindberg-Yücel and Bage, 2013).

Diabetes mellitus (DM) is a cluster of metabolic disorders that are chiefly characterized by hyperglycemia due to impaired secretion or action of insulin, or both. The mainstream of cases of diabetes falls into two wide etiopathogenetic classes.  Type 1 diabetes, which comprises only 5–10% of those having diabetes, the cause of which is an absolute deficiency of insulin secretion due to an autoimmune process.  The most common form of diabetes is type 2 diabetes mellitus (T2DM), which accounts for nearly 90-95% of all diabetic patients and is due to a combination of insulin resistance and an insufficient compensatory insulin secretion response (American Diabetes Association, 2013).

For decades, a significant close association between diabetes and periodontitis has been confirmed, where evidence suggests that diabetes is a contributing factor in worsening the condition of periodontal disease, on the same hand, periodontal disease has a higher incidence in diabetics, and is claimed to be more prevalent and severe in diabetics when compared to healthy individuals (Grossi and Genco, 1998,  Papapanou, 1996).

Coexistence of periodontitis in diabetic patients is attributed to failure of the body's  immune system to completely eliminate the source of inflammation such as microorganisms, this in turn, keeps the inflammatory process continuously activated, therefore, a chronic inflammatory reaction is induced( Herring and Shah, 2006). This chronic inflammatory response is the main etiologic factor of systemic upregulation of different pro-inflammatory cytokines such as interleukins (IL-1β, 4, 6, 8, and 10) as well as tumor necrosis factor-α (TNF-α) (Grossi and Genco, 1998, Iacopino, 2001, Sun et al., 2010, Correa et al, 2010).

Moreover, these pro-inflammatory cytokines are blamed to be a great player in inducing insulin resistance, initiating pancreatic beta-cell destruction, and dis-regulating lipid metabolism, leading to hyperlipidemia with both low-density lipoproteins and triglycerides (Iacopino, 2001, Sun et al., 2010).

Adipokines are cytokines not only derived from adipocytes but also from endothelial cells, fibroblasts  and various amounts of immune cells as macrophages, lymphocytes, granulocytes, mast cells.  Adipokines regulate important biological processes in target organs such as the brain, liver, skeletal muscle, cardiovascular and immune systems ( Feve et al.,2016) Insulin resistance is associated with an excessive  metabolic abnormalities including dyslipidemia, low grade inflammation and altered adipokine levels and many of these abnormalities can directly or indirectly accelerate the malfunctioning of glucose metabolism (Kumari et al., 2019).

Chemerin is one of the adipokines, which was first discovered as a chemoattractant peptide that believed to cause migration of macrophages, leukocytes, and dendritic cells all with ChemR23 receptors to the site of inflammation. (Monnier et al., 2018). Although the white adipose tissue is described as the main source of chemerin, later on, a high concentration was also identified in the liver (Döcke et al., 2013 and Weigert et al., 2010), lower lungs, brown adipose tissue, heart, ovaries, kidneys, skeletal muscle, and pancreas (Rourke et al., 2013).

It was demonstrated that chemerin is mostly associated with a variety of chronic inflammatory conditions, such as diabetes, rheumatoid arthritis and cardiovascular diseases (Huang et al., 2015). In addition, circulating chemerin levels was also correlated positively with TNF-α, IL-6 and C-reactive protein (CRP), resistin and leptin which are suggested as markers of inflammation. (Lehrke M et al, 2009, Weigert et al., 2010) Therefore, systemic chemerin is mainly related to inflammation rather than obesity in type 2 diabetes. Consequently, Chemerin is associated with markers of inflammation and components of the metabolic syndrome (Lehrke et al., 2009).

Fibroblast growth factor 21 (FGF21) is another adipokine which  is a 181 amino acid (w20 kDa) circulating protein that is derived from a 209-amino acid mature protein encoded by the FGF21 gene which is located in chromosome 19 (Nishimura et al., 2000), FGF21 is found to belong to the human FGF superfamily, which was initially named according to its ability to stimulate fibroblast proliferation (Long and Kharitonenkov, 2011)

FGF21 is found to be expressed in several tissues such as liver (Nishimura et al., 2000), pancreas (Johnson et al., 2009), and adipocytes (Zhang et al., 2008). It is demonstrated to act as a metabolic regulator as it applies a variety of pharmacological effects on metabolism (Itoh, 2014).  Moreover, circulating FGF21 levels are found to be elevated in various metabolic diseases, such as obesity, insulin resistance, and type 2 diabetes mellitus (Zhang et al., 2008, Mraz et al., 2009)

FGF21 is blamed to play various actions in energy metabolism and injury protection as a hepatokine, adipokine, and myokine in an endocrine or autocrine/paracrine manner (Itoh, 2014). Although of the several metabolic effects of FGF2, whether its action is beneficial or detrimental in the physiological or pathological conditions are still unclear (Tezze et al., 2019).

Various inflammatory markers in GCF have been detected to play a significant role in the disease mechanism of both periodontitis and type 2 DM (Pradeep et al., 2013). Therefore, the present study aimed to spot the light on the possible role of crevicular levels of both FGF21 and chemerin as potential biomarkers of periodontitis in diabetic and non-diabetic patients in an effort to understand their role in the link between both diseases.

Subjects and Methods:

This clinical trial has been registered at Clinical Trials.gov (identifier NCT04012983) and has been approved by the research ethics committee of the faculty of Dentistry, Cairo University by the number 19632 in 25-6-2019.

The present study was conducted on forty five individuals (ranging in age from 35-60 years) recruited from the outpatient clinic of Oral Medicine, and Periodontology Department, Faculty of Dentistry, Cairo University in the period from Mar 2018 to September 2018.

The participants were divided into the following three groups:

Group (1): 15 patients with periodontitis and controlled t2DM and no other systemic disease.

Group (2): 15 systemically healthy individuals having periodontitis.

Group (3): 15 periodontally  and systemically healthy individuals.

Medical history for all participants was acquired according to the modified Cornell medical index (Abramson, 1966), and an informed consent was signed by all participants after explaining the nature and objectives of the study.

Glycated hemoglobin A1c (HbA1c) and fasting plasma glucose (FPG) measures were used to verify all patients' glycemic condition. At the start of the investigation, patients with DM eligible to participate in the study were determined according to the American Diabetes Association 2010, and had to exhibit HbA1c value of  6-8% and FPG ≥126 mg/dL. In order to verify the non-diabetic status of healthy individuals their HbA1c was shown to be <6% and FPG < 126 mg/dL.

Inclusion criteria

All participants were selected with age ranging between 35 and 60 years with a minimum of 20 natural teeth. Controlled type 2DM patients exhibiting HbA1c 6-8% and FPG ≥126 mg/dL for more than one year who did not complain of any systemic diseases other than t2DM.

As for periodontitis patients in groups 1 and 2, and according to the American academy of periodontology 2000,participants with moderate to severe periodontitis having gingival index GI ≥ 1, probing depth PD ≥ 5, and clinical attachment level CAL ≥ 4 mm, and bone loss affecting > 30% of existing teeth on clinical/radiographic examination were included in the present investigation.

Exclusion criteria

Systemic diseases such as, uncontrolled diabetes mellitus, human immune-deficiency virus, cancer, or any other diseases which may affect the biomarkers levels and the periodontal conditions. Exposure to steroid therapies, radiation/immune-suppressive therapies, allergic reaction to any kind of drug. Smoking over the past 5 years.

Periapical pathologies, exposure to mechanical force as a result of occlusion/ orthodontics. History of either periodontal or drug therapies within the previous 6 months, as antibiotic therapy, anti-inflammatory drugs, or other pharmacological treatments.

Periodontal examination:

All participants were subjected to a full- mouth examination. As for periodontitis patients, clinical parameters were recorded, including plaque index (PI), gingival index (GI), probing depth (PD), and clinical attachment level (CAL). PD was measured from the gingival margin to the base of the periodontal pocket, and CAL was measured from the cemento-enamel junction to the base of the periodontal pocket. All of these readings were recorded at six sites (mesio-buccal, mesio-lingual, mid-buccal, disto-buccal, distolingual, and mid-lingual) for all teeth.

Clinical parameters were recorded using a Williams periodontal probe (Hu-Friedy, Chicago, IL, USA) by the same researcher (EA). To ensure intra-examiner reproducibility, individuals were evaluated twice with a 48-h interval. A difference falling within a < 10% mm range between the two measurements, was considered to be acceptable (Schwarz et al., 2006).

GCF Samples collection:

For all groups, samples were assembled the day after the patients had been given clinical evaluation to evade mixing GCF with blood accompanying probing of inflamed areas. For each individual in group 1 and 2, two sites were chosen for sampling. GCF samples were obtained from mesio-buccal or disto-buccal sites in teeth with the most severe attachment loss. Before GCF sampling, supra-gingival plaque was removed from the sampling site by means of a sterile cotton roll. The site was then washed with water, secluded with cotton balls, and slowly air-dried to avoid saliva contamination. Paper strips were then located inside the crevice until a small amount of resistance was met and then remained in place for 30 seconds. This procedure was performed carefully to prevent damage to gingival tissues manually. Any blood and/or saliva contaminated samples were discarded. As for the healthy control group, samples were acquired from the upper first molar. From each study participant, two strips were put into a single (coded) Eppendorf container, then combined to form a single sample, and quickly stored at -80C until they could be evaluated.

Measurement of chemerin level in GCF:

The level of chemerin was measured using the RD191136200R Human Chemerin ELISA which is a sandwich enzyme immunoassay for the quantitative measurement of human chemerin. The kit was provided by BioVendor – Laboratorní medicína, Guang Zhou, CHINA. In the Biovendor Human Chemerin ELISA, standards, quality controls and samples are incubated in microtitration wells pre-coated with polyclonal anti-human chemerin antibody. After a 60 minute incubation followed by washing, the biotin labelled polyclonal anti-human chemerin antibody is added and incubated with the captured chemerin for 60 minutes. After another washing, streptavidin-HRP conjugate was added. Following 30 minutes incubation and the final washing step, the remaining conjugate was allowed to react with the substrate solution (TMB). Acidic solution was then added to stop the reaction and then the absorbance of the resulting yellow product was measured. The absorbance is proportional to the concentration of chemerin. A standard curve then was constructed by plotting absorbance values against chemerin concentrations of standards. Using this standard curve, the concentrations of unknown samples are determined.

Quantitation of Human fibroblast growth factor 21 in GCF:

Human fibroblast growth factor 21 was measured in all participants using Human Fibroblast growth factor 21 ELISA Kit which was provided by Bioassay Technology Laboratory, Shanghai, China.  This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). FGF-21 was added to the wells pre-coated with FGF-21 monoclonal antibody. After incubation, the biotin-conjugated anti-human FGF-21 antibody was added to bind to human FGF-21. After incubation, unbound biotin-conjugated anti-human FGF-21 antibody was washed away during a washing step. Streptavidin-HRP was added to bind to the biotin-conjugated anti-human FGF-21 antibody. After incubation, unbound Streptavidin-HRP was washed away during a washing step. Substrate solution was then added and colour develops in proportion to the amount of human FGF-21. The reaction was terminated by addition of acidic stop solution and absorbance was measured at 450 nm.

Sample size calculation:

Sample size calculation was done based on the previous papers by Wang et al.,2017 and Šimják  et al.,2018; that reported the difference in FGF-21 level between diabetic patients with periodontitis and healthy controls to be 100±70 pg/mL. 14 patients were needed to detect that difference using power 95% and 5% significance level. Sample  size  calculation  was achieved  using  PS:  Power  and  Sample  Size  Calculation  software  Version  3.1.2  (Vanderbilt University, Nashville, Tennessee, USA).

Statistical analysis:

Qualitative data were presented as frequencies (n) and percentages (%). Numerical data were presented as mean, median, standard deviation (SD), minimum and maximum values. Kruskal Wallis Test was used to test the difference between the three groups in term of Chemerin (ng/ml), and FGF21 (pg/ml) levels and Post-hoc analysis using Mann-Whitney test was performed. Diagnostic performance of different markers was done by Roc curve. All p-values are two-sided. P-values < 0.05 were considered significant

Statistical analysis was performed with IBM^® SPSS^® Statistics Version 20 for Windows.

Results:

The study was conducted on forty five individuals (22 males (48.9%) and 23 females (51.1%) ranging in age from 35-60 years with mean age 43.5 years and were divided into 3groups

In terms of the clinical parameters in periodontitis patients with or without diabetes,there was no significant difference in all clinical parameters PI, GI, PD, CAL, between the two groups with P values, 0.6766, 0.3122, 0.4376, and 0.2480 respectively. (p<0.05)