|Year : 2019 | Volume
| Issue : 2 | Page : 110-115
“Estimation of Midkine Levels in Gingival Crevicular Fluid and Serum in Periodontal Health, Disease and After Treatment” − A Clinico Biochemical Study
Dandu Siva Sai Prasad Reddy1, Vemuri Vineetha1, Dodla Alekya1, M. D. Sameevulla1, Nagireddy Ravindra Reddy1, D. S.Madhu Babu2
1 Department of Periodontics, CKS Theja Institute of dental sciences, Tirupathi, Andhra Pradesh, India
2 Department of Dental Surgery, Sri Padmavathi Medical College for women, SVIMS, Tirupathi, Andhra Pradesh, India
|Date of Submission||06-Dec-2019|
|Date of Decision||10-Dec-2019|
|Date of Acceptance||11-Dec-2019|
|Date of Web Publication||29-Jan-2020|
D. S.Madhu Babu
Department of Dental Surgery, Sri Padmavathi Medical College for women, SVIMS, Tirupathi, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: The objective of the present study was to evaluate the role of Midkine in periodontal disease progression and also to investigate the effect of periodontal therapy on Midkine concentration in serum and gingival crevicular fluid (GCF). Materials and Methods: Clinical parameters including gingival index, pocket probing depth, clinical attachment level were recorded for 60 subjects divided into four equal groups Group I (healthy), Group II (gingivitis), Group III (chronic periodontitis), and Group IV (post treatment group). Scaling and root planning were performed and GCF and serum were collected initially and after 8 weeks of treatment. Midkine levels were estimated using enzyme-linked immunosorbent assay. Results: The mean Midkine concentration in GCF and serum was found to be the highest in group III, and significantly defers from group-I, II, and IV. The results of present study also suggest that Midkine levels increased progressively in GCF and serum from healthy to periodontitis subjects and levels decreased considerably after scaling and root planning. Conclusion: As the periodontal disease progresses, there is a substantial increase of Midkine concentrations in serum and GCF. The data indicate that high GCF and serum levels of Midkine are at significantly greater risk for progression of periodontitis However, controlled, longitudinal studies are needed to confirm this possibility.
Keywords: Gingival crevicular fluid, Midkine, scaling and root planning, serum
|How to cite this article:|
Reddy DS, Vineetha V, Alekya D, Sameevulla MD, Reddy NR, Babu DS. “Estimation of Midkine Levels in Gingival Crevicular Fluid and Serum in Periodontal Health, Disease and After Treatment” − A Clinico Biochemical Study. J Orofac Sci 2019;11:110-5
|How to cite this URL:|
Reddy DS, Vineetha V, Alekya D, Sameevulla MD, Reddy NR, Babu DS. “Estimation of Midkine Levels in Gingival Crevicular Fluid and Serum in Periodontal Health, Disease and After Treatment” − A Clinico Biochemical Study. J Orofac Sci [serial online] 2019 [cited 2020 Jul 13];11:110-5. Available from: http://www.jofs.in/text.asp?2019/11/2/110/276717
| Introduction|| |
Periodontitis is a bio film-induced chronic inflammatory disease that leads to the destruction of the periodontium, that is the tooth-supporting structures such as the gingiva and underlying alveolar bone. Tooth-associated bio film or dental plaque is required, but not sufficient to induce periodontitis because it is the host inflammatory response to this microbial challenge that ultimately can cause destruction of the periodontium. 
Host response has traditionally been considered to be mediated mainly by B and T-lymphocytes, neutrophils, and monocytes/macrophages. These are triggered to produce inflammatory mediators, including cytokines, chemokines, arachidonic acid metabolites, and proteolytic enzymes, which collectively contribute to tissue degradation and bone resorption by activation of several distinct host degradative pathways. 
Chemokines are synthesized by several cell types, including endothelial, epithelial, and stromal cells, as well as leukocytes. The selective production of chemokines may be involved in the determination of the spatial localization of the inflammatory cells in periodontal tissues for optimization of host defenses, and may contribute to leukocyte infiltration into the infected and inflamed area, thus limiting tissue damage. 
Midkine (MK) is a retinoic acid-inducible heparin-binding cytokine. MK is structurally unrelated to fibroblast growth factors, typical heparin binding growth factors, and is the initial member of a new cytokine/growth factor family, which so far has only two members, MK and pleiotrophin. Since its discovery, MK has been implicated in the regulation of embryogenesis, based on the finding that it is expressed in various tissues in a manner strictly controlled both spatially and temporally during embryogenesis. In a healthy adult, MK is expressed only in highly restricted sites. Recently, it was found that an anti-MK antibody inhibits the differentiation of the tooth germ in vitro. 
MK expression is induced in damaged tissues, especially after ischemia in blood vessels, the brain cortex and the myocardium, and exhibits two effects, an enhancement of inflammation and a promotion of survival and repair. Thus, MK is either beneficial or harmful to the injured tissue, depending on its origin. MK enhances inflammation by promoting the migration of inflammatory leukocytes, inducing synthesis of chemokines and suppressing the increase of regulatory T cells. 
MK was found to be rarely expressed in the adult organism, but different types of cancer cells showed high MK expression associated with a poor prognosis of the patients. However, there is growing evidence that MK may also play an important role in chronic inflammatory disorders including kidney diseases, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. 
To date, there is no study reporting the inflammatory role of MK in periodontal tissues and its concentrations in GCF and serum in periodontal health and disease.
Thus, in view of the aforementioned findings, this clinico-biochemical study was undertaken to estimate the MK levels in GCF from subjects with clinically healthy periodontium, gingivitis, and chronic periodontitis and in addition to explore the effectiveness of periodontal interventional therapy on GCF levels of MK in subjects with chronic periodontitis as to get a more detailed insight into its possible role in initiation and progression of periodontal disease.
| Materials and Methods|| |
The study population consisted 60 subjects (31 women and 29 men) aged 22–48 years, who were selected from the outpatient department. The potential benefits or detrimental effects were discussed with the study subjects, and written informed signed consent was obtained from the participants who agreed to participate voluntarily. Ethical approval for this study (Ethical Committee Ref no:139/IEC-CKS /Lr/17) was provided by the Ethical Committee IEC of CKS, Tirupati, on 15 December, 2016. Inclusion criteria included individuals aged 22-48 years, with no history of previous periodontal treatment within the preceding six months and with at least ≥ 20 natural teeth. Exclusion criteria included systemic diseases, such as diabetes, hypertension, heart diseases, rheumatoid arthritis, and respiratory diseases, that affected PD progression, antiinflammatory drug and antibiotics use, any periodontal treatment in the preceding six months, pregnancy, and lactation.
Periodontal classification provided by American Academy of Periodontology was used to divide subjects into groups containing subjects with Chronic Periodontitis and Gingivitis and healthy control subjects. 
- Group I (Healthy): Twenty subjects with clinically healthy periodontium. Gingival Index (GI) = 0, Probing Pocket Depth (PPD) = ≤3 mm, Clinical Attachment Level (CAL) = 0, and no evidence of bone loss on radiographs.
- Group II (Gingivitis): Twenty subjects with clinical signs of gingival inflammation. GI = >1, PPD = ≤3 mm, CAL = 0, and no evidence of bone loss on radiographs.
- Group III (Chronic Periodontitis): Twenty subjects with signs of clinical inflammation. GI = >1, PPD = ≥5 mm, CAL = ≥3 mm, and radiographic evidence of bone loss at more than 10 sites.
- Group IV (post-treatment Group): Patients in Group III were treated with Scaling and Root planing (SRP). The GCF samples were collected from the same site 8 weeks after SRP.
The participants were subjected to clinical examinations for the following periodontal clinical parameters: GI, PPD, and CAL. A single examiner recorded all measurements from six sites for all teeth by using a UNC-15 periodontal probe to ensure adequate intra-examiner reproducibility. Marginal gingival bleeding was recorded using GI. PPD and CAL were measured from a fixed reference point on an acrylic stent and from the Cemento-Enamel Junction. The GI, PPD, CAL, and MK level assessments in GCF and serum were conducted at the baseline and 8 weeks after SRP.
Collection of GCF
First examiner conducted all clinical and radiological examinations, group allocations, and sampling site selection. Samples were collected on the subsequent day by a second examiner to ensure the masking of the sampling examiner and to avoid the contamination of GCF with blood-associated probing at the inflamed sites. In this study, we selected only one site each in Group II and Group III, whereas in Group I, multiple sites without inflammation were selected to collect adequate GCF. In Group II, the site with the highest clinical signs of inflammation was selected for GCF collection. In Group III, the site with the highest clinical signs of inflammation, CAL, and radiographic bone loss was selected for GCF collection.
On the following day of the clinical examination, the identified site was isolated using a cotton roll and saliva ejector to prevent salivary contamination. The site was gently air-dried, and clinically detectable supragingival plaque was removed using a curette without touching the marginal gingiva. GCF was collected by placing a micropipette at the entrance of the gingival sulcus. A standardized volume of 1 μl GCF was collected from each site after calibrating white color-coded 1–5 μl-calibrated volumetric micropipettes (Sigma Aldrich). Group III received SRP during the same appointment, after GCF collection. After 8 weeks, GCF was collected from same site. The collected GCF samples were placed immediately into individual microcentrifuge tubes containing 300 μl of phosphate-buffered saline. The samples were stored at −70°C until the Enzyme-Linked Immunosorbent Assay (ELISA) was performed. During the 8 weeks, subjects were seen at 1-week intervals, and plaque control measures were performed.
Collection of serum
Blood (5 ml) was collected from antecubital fossa through venipuncture by using a 20-gauge needle with a 5-ml syringe and was immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature, and the Serum was separated from the blood after 1 hr by centrifuging the blood at 3000 ×g for 10 min. The serum was immediately transferred to a plastic vial and stored at −70°C until ELISA was performed.
Determination of MIDKINE in GCF and serum
MK levels in 160 GCF and Serum samples were determined using solid-phase sandwich ELISA (catalog no. RHF911CKC, Antigenix America Inc., USA). The samples were run in triplicate to ensure accuracy and to provide sufficient data for the statistical validation of the results. An ELISA reader (Biorad, USA) with primary and reference wavelengths of 450 and 655 nm, respectively, was used to measure the absorbance of the substrate. The MK levels in the tested samples were evaluated using a standard curve plot for which the absorbance values of standards were provided along with the kit. The absorbance readings were converted into definite volumes (ng/μl) by using a standard reference curve.
Descriptive Statistical Analysis
Data were analyzed using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). A sample size of 20 was sufficient to achieve more than 80% power at a 0.1 level of significance. Group comparisons for nonparametric variables were conducted using the Kruskal–Wallis Test. In addition, pair wise comparisons were conducted using the Mann–Whitney U Test to determine the pair(s) that differed. The statistical significance of the MK levels before and after SRP was analyzed using the Wilcoxon Signed-Rank Test.
| Results|| |
All the samples in each group tested positive for the presence of MK. The mean GCF and serum concentration was highest in group-III. The mean GCF and serum MK concentration in group-IV was found to lie between group II and III [Table 1] and [Table 2].
|Table 1 Mean GCF concentration of Midkine of Group I, Group II, Group-III and Group IV|
Click here to view
|Table 2 Mean serum concentration of Midkine of Group I, Group II, Group III, and Group IV|
Click here to view
The Kruskal–Wallis and Mann-Whitney U tests were carried out to determine whether there any significant differences in GCF and serum MK levels between the study groups as shown in [Table 3] and [Table 4]. The results indicate that MK both in GCF and serum increase progressively from healthy to periodontitis patients.
|Table 3 Kruskal–Wallis test comparing mean Midkine concentration in GCF and serum between four groups|
Click here to view
|Table 4 Pair wise comparison using Mann-Whitney U test for GCF and serum MIDKINE|
Click here to view
When group-III and group-IV were compared using the Wilcoxon signed rank test, as shown in the [Table 5], the difference in concentrations of MK in GCF and serum was statistically significant (P < 0.05), indicating that after SRP, the mean concentrations of MK in GCF and serum decreased considerably in accordance with a decrease in CAL.
|Table 5 Wilcoxon signed rank test to compare Midkine concentration in GCF and serum Group III and Group IV|
Click here to view
Confidence interval was calculated for differentiating the limits of GCF and Serum MK values in different groups to consider MK as inflammatory biomarker [TABLE 6].
|TABLE 6 Differentiating values for different groups for GCF and serum Midkine (ng/µl)|
Click here to view
| Discussion|| |
The incidence and progression rate of periodontal disease depends on complex interaction between periodontopathic bacteria and cells of the host immune system. These interactions are mediated by cytokines and chemokines, which are produced by both resident and emigrant cells at the site of inflammation. Cells that produce cytokines include macrophages/monocytes, dendritic cells, lymphocytes, neutrophils, endothelial cells, and fibroblasts. Cytokines are central for the pathogenesis of an ever increasing number of diseases, including periodontal disease. 
MK and pleiotrophin (PTN) comprise a distinct family of heparin-binding growth factors that are multifunctional, acting both as growth factors and activators of neutrophils and also both MK and PTN have antibacterial activity against a wide variety of bacteria. 
MK participates in inflammatory processes by exerting two different activities. Firstly, MK enhances the migration of neutrophils and macrophages both, by direct action of MK and by inducing chemokine expression. Secondly, MK suppresses differentiation of regulatory T-cells partly by inhibiting the development of tolerogenic dendric cells, which are essential for the differentiation. Inflammation, which is enhanced by MK is part of the immune system. MK also has anti-microbial activities and is regarded as a component of the innate immune system. Furthermore, MK has been found to enhance the survival of B cells. Thus, MK plays diverse roles in counteracting infection upon injury. 
A study done by Keisuke Hatori et al. detected MK expressed by inflammatory cells, such as macrophages, lymphocytes and neutrophils, as well as by endothelial cells in periapical granulomas but not in healthy gingival tissues. However, till today there are no studies which have investigated MK concentrations in GCF and correlated them to that of Serum MK concentrations in periodontal health, disease and after treatment. The present study is thus first study designed to investigate the concentrations of MK in GCF and Serum in periodontal health and disease and also to assess the role of periodontal therapy on MK concentrations.
In previous studies, the expression of MK has been noted in several systemic inflammatory diseases in humans. Ludwig et al. conducted a study to demonstrate that MK was critically involved in recruitment of PMNs during acute inflammation, playing a key role for adhesion, and subsequent extravasation. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of β2 integrins, thereby facilitating PMN trafficking during acute inflammation. Takada et al. conducted a study to examine the MK expression in the inflammatory synovitis of rheumatoid arthritis and osteoarthritis, MK was detected in synovial fluid, synoviocytes, and endothelial cells of new blood vessels. Normal synovial fluid and non-inflammatory synovial tissue did not contain detectable MK. Hiroshi Narita et al. conducted a study to demonstrate that MK is expressed by infiltrating macrophages in in-stent restenosis in hypercholesterolemic rabbits. The results suggested that macrophages are the major source of MK in the atherosclerotic neointima. Malgorzata Krzystek-Korpack et al. conducted a study to assess the concentrations of circulating MK in patients with ulcerative colitis with respect to disease activity and severity of inflammatory response. The study concluded that UC is associated with increased circulating MK, which corresponds with clinical, endoscopic inflammatory and angiogenic activity and also showed that MK as a marker of UC was comparable to that of CRP (C-Reactive Protein).Vahid Shaygannejad et al. conducted a study to demonstrate the correlation between MK Serum levels and concentration of pro and anti- inflammatory cytokines in Multiple Sclerosis (MS) patients. The results of this study showed that the MK concentration in MS patients is lower than healthy controls.
The diagnosis of active phases of periodontal disease and the identification of patients at risk for active disease represent a challenge for both clinical investigators and clinicians. Advances in oral and periodontal disease diagnostic research are moving towards methods whereby periodontal risk can be identified and quantified by objective measures, such as biomarkers. These biomarkers of host response can be found in gingival crevicular fluid, saliva and serum samples and can potentially be used as diagnostic markers. 
Earlier studies used filter paper strips and Periotron 8000 and 6000 which can result in non-specific attachment of the analyte to filter paper fibers ensuing in a false reduction in the detectable MK levels, which underestimates the correlation of levels MK to disease. In the present study, the extracrevicular (unstimulated) method of GCF collection using microcapillary pipettes is done to ensure atraumatism, to obtain an undiluted sample of native GCF, the volume of which could be accurately assessed, and to avoid non-specific attachment of the analyte to filter-paper fibers. ,
Periodontal infections are not only localized to the marginal periodontium, but also patients present in systemic inflammation, that was indicated by elevated serum levels of various inflammatory markers when compared to those in unaffected control populations.  GCF is a serum transudate that is enriched with microbial and host products that arise as a result of the current inflammatory dynamics of the host–biofilm interaction.  In the present study both GCF and Serum were evaluated for the change in the level of MK between health and diseased individuals.
In the present study MK concentrations in GCF and serum were analyzed by ELISA. In the present study the mean concentrations of MK in GCF and serum were found to increase proportionately from health to periodontitis. while in gingivitis the mean concentrations of MK fell between healthy and periodontitis groups. These levels increased proportionally with the severity of disease in Group II and III showing positive correlation with clinical parameters. When the Chronic Periodontitis patients were treated by non-surgical periodontal therapy (SRP) with strict oral hygiene measures, the mean concentration of MK in GCF and serum reduced after treatment. This decrease in MK concentration further correlated positively with the decrease in scores of clinical parameters, suggesting its association with the severity of disease. The possible reason for increase in GCF levels of MK in this study could be because of migration of neutrophils and macrophages both by direct action of MK and by inducing chemokine expression. The positive correlation between clinical parameters and MK level in GCF could be attributed to the release of chemokines at tissue injury site. The possible reason for increase in Serum levels of MK could be either spill over from the GCF or gingival tissues to peripheral circulation or it could be due to systemic inflammatory response to progressive disease in the periodontal pocket.
The results of the present study indicate that the concentration of MK in GCF and Serum increased proportionately with the severity of disease. The proportionately increase in levels from healthy to gingivitis to periodontitis groups further confirmed that MK was actively secreted by the predominant cells of periodontal disease activity.The variability of MK concentrations within patients of each group could be attributed to the role of MK in different stages of disease process at the time of collection of GCF and Serum samples. The high concentration of MK in two participants (0.177 ng/µl, 0.135 ng/µl in serum) in the Healthy Group could have been due to the subclinical inflammation or allergy or any infection not reported by patients. Low MK levels were found in two GCF sample of patients with periodontitis (0.060 ng/µl, 0.108 ng/µl)and two serum samples (0.100 ng/µl, 0.129ng/µg) which may be because this diseased site was probably stable. The wide range observed in the levels of MK in Gingivitis and Periodontitis could result, in part, from differences in disease activity and crevicular fluid flow at the time of collection, as well as from variations in the number of defense cells migrating into the crevice.
| Conclusion|| |
In conclusion, within the limitation of our study, the data indicates that MK in GCF and serum shows dynamic changes according to severity of periodontal disease and the levels of MK concentrations have stronger relationship with clinical parameters. It can be used as a marker of gingival inflammation in order to determine the effect of periodontal therapy. However, further longitudinal studies are needed to evaluate the concentrations of MK in the periodontal disease tissues and GCF will be beneficial in clarifying the role in the pathogenesis of periodontitis and to validate MK as a “Novel Biomarker” of periodontal disease progression.
| Financial support and sponsorship|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hajishengallis G. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends Immunol 2014;35:3-11.
Yucel-Lindberg T, Båge T. Inflammatory mediators in the pathogenesis of periodontitis. Expert Rev Mol Med 2013;15:e7.
Silva TA, Garlet GP, Fukada SY, Silva JS, Cunha FQ. Chemokines in oral inflammatory diseases: apical periodontitis and periodontal disease. J Dent Res 2007;86:306-19.
Takada T, Toriyama K, Muramatsu H, Song XJ, Torii S, Muramatsu T. Midkine, a retinoic acid-inducible heparin-binding cytokine in inflammatory responses: chemotactic activity to neutrophils and association with inflammatory synovitis. J Biochem 1997;122:453-8.
Muramatsu T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 2010;86:410-25.
Weckbach LT, Muramatsu T, Walzog B. Midkine in inflammation. Scientific World Journal 2011;11:2491-505.
Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6.
Pradeep AR, Hadge P, Chowdhry S, Patel S, Happy D. Exploring the role of Th1 cytokines: interleukin-17 and interleukin-18 in periodontal health and disease. J Oral Sci 2009;51:261-6.
Nordin SL, Sonesson A, Malmsten M, Mörgelin M, Egesten A. The epithelium-produced growth factor midkinehas fungicidal Properties. J Antimicrob Chemother 2012;67:1927-36.
Muramatsu T. Structure and function of Midkine as the basis of its pharmacological effects. Br J Pharmacol 2014;171:814-26.
Hatori K, Takeichi O, Ogiso B, Maeno M, Komiyama K. Midkine expression in human periapical granulomas. J Endod 2011;37:781-5.
Weckbach LT, Gola A, Winkelmann M, Jakob SM, Groesser L, Borgolte J. et al.
.. The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via β2integrins (CD11/CD18). Blood 2014;123:1887-96.
Narita H, Chen S, Komori K, Kadomatsu K. Midkine is expressed by infiltrating macrophages inin-stent restenosis in hypercholesterolemic rabbits. J Vasc Surg 2008;47:1322-9.
Krzystek-Korpacka M, Neubauer K, Matusiewicz M. Clinical relevance of circulating midkine in ulcerative colitis. Clin Chem Lab Med 2009;47:1085-90.
Shaygannejad V, Montazeri S, Jamshidian A, Tahani S, Gharagozloo M, Ashtari F. et al.
.. Correlation of midkine serum level with pro- and anti-inflammatory cytokines in multiple sclerosis. Iran J Immunol 2014;11:134-8.
de Queiroz AC, Taba M Jr, O’Connell PA, da Nóbrega PB, Costa PP, Kawata VK. et al.
.. Inflammation markers in healthy and periodontitis patients. A preliminary data screening. Braz Dent J 2008;19:3-8.
Pradeep AR, Daisy H, Hadge P, Garg G, Thorat M. Correlation of gingival crevicular fluid interleukin-18 and monocyte chemoattractant protein-1 levels in periodontal health and disease. J Periodontol 2009;80:1454-61.
Sueda T, Bang J, Cimasoni G. Collection of gingival fluid for quantitative analysis. J Dent Res 1969;48:159.
Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ, De Nardin E. Periodontal infections contribute to elevated systemic C-reactive protein level. J Periodontol 2001;72:1221-7.
Lamster IB, Ahlo JK. Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Ann N Y Acad Sci 2007;1098:216-29.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [TABLE 6]