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ORIGINAL ARTICLE
Year : 2018  |  Volume : 22  |  Issue : 1  |  Page : 40-44  

Effect of scaling and root planing on gingival crevicular fluid level of YKL-40 acute phase protein in chronic periodontitis patients with or without type 2 diabetes mellitus: A clinico-biochemical study


Department of Periodontics, Bapuji Dental College and Hospital, Davangere, Karnataka, India

Date of Submission28-Apr-2017
Date of Acceptance05-Feb-2018
Date of Web Publication28-Feb-2018

Correspondence Address:
Dr. Sini Damodar
Department of Periodontics, Bapuji Dental College and Hospital, Davangere - 577 004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisp.jisp_95_17

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   Abstract 


Background: The aim of the present study was to evaluate the gingival crevicular fluid (GCF) levels of YKL-40 acute phase protein in chronic periodontitis (CP) with and without type 2 diabetes and also to assess the effect of periodontal therapy (scaling and root planing [SRP]) on this GCF biomarker and the clinical parameters. YKL-40 is derived from tyrosine (Y), lysine (K), and leucine (L) with a molecular weight of 40 kDa. Materials and Methods: A total of 105 individuals (30–60 years) were grouped as 35 individuals each in three groups (Group I – healthy; Group II – CP with diabetes mellitus [DM]; and Group III – CP). Clinical parameters including plaque index, gingival index, probing pocket depth, and clinical attachment level followed by GCF sample collection from test sites were done at baseline and 6 weeks after SRP (among Group II and Group III patients). GCF YKL-40 level was determined by enzyme-linked immunosorbent assay. Results: The mean GCF YKL-40 level at baseline was significantly lower for Group I (309.81 ± 124.93 pg/ml) as compared to Group II (924.88 ± 415.28 pg/ml) and Group III (834.08 ± 270.42 pg/ml), respectively (P < 0.001). The level reduced significantly 6 weeks after SRP for Group II (507.6 ± 265.03 pg/ml) and Group III (499.54 ± 293.38 pg/ml) (P < 0.001). Conclusion: The level of GCF YKL-40 in CP patients with or without DM is higher than healthy individuals and the level reduced 6 weeks post-SRP among Group II and Group III. Hence, YKL-40 can be considered as an important biomarker in the diagnosis of CP.

Keywords: Biomarkers, chronic periodontitis, diabetes mellitus, gingival crevicular fluid


How to cite this article:
Damodar S, Mehta DS. Effect of scaling and root planing on gingival crevicular fluid level of YKL-40 acute phase protein in chronic periodontitis patients with or without type 2 diabetes mellitus: A clinico-biochemical study. J Indian Soc Periodontol 2018;22:40-4

How to cite this URL:
Damodar S, Mehta DS. Effect of scaling and root planing on gingival crevicular fluid level of YKL-40 acute phase protein in chronic periodontitis patients with or without type 2 diabetes mellitus: A clinico-biochemical study. J Indian Soc Periodontol [serial online] 2018 [cited 2019 Jan 22];22:40-4. Available from: http://www.jisponline.com/text.asp?2018/22/1/40/226374




   Introduction Top


It is widely recognized that periodontitis is one such condition affecting millions of people every year around the world. Undoubtedly, there are immeasurable components of virulent factors, host response, and environmental influences, which are involved and play an important role in some aspects of periodontal pathogenesis. However, not all associated components are important for causing disease.[1] Acute phase proteins (APPs) are generated in the body by acute phase reaction/response (APR), which is the systemic reaction of microbes to any injury, infection, wound, and malignancy. Some of the widely studied APPs are C-reactive protein (CRP), haptoglobin, fibrinogen, fibronectin, and transferrin. However, the most recent addition to the class of APPs is the YKL-40.[2]

YKL-40 also known as human cartilage glycoprotein-39 (HC gp-39) is principally derived from neutrophils, macrophages, endothelial cells, osteoclasts, and osteocytes.[3] According to the various authors, Hakala et al, Rathcke and Vestergaard the name YKL-40 is derived from three amino acids, i.e., tyrosine (Y), lysine (K), and leucine (L) at the N terminal with apparent molecular weight of 40 kDa.[3],[4] Although the complete biological functions of YKL-40 are not known, literature has revealed that increased level of YKL-40 is seen in certain systemic conditions such as diabetes mellitus (DM), sarcoidosis, inflammatory bowel disorder, systemic lupus erythematosus, liver fibrosis, arthritis, atherosclerosis, cancers, coronary artery disease, chronic obstructive pulmonary disorder, emphysema, and Alzheimer's disease.[5],[6],[7],[8],[9]

DM is a heterogeneous group of metabolic disorder characterized by altered level of blood sugar. According to the International Diabetes Federation South East Asia, 415 million people have diabetes in the world, and 69.1 million cases of diabetes were reported in India in 2015. Periodontitis has been designated as the sixth complication of diabetes by Harald Loe. DM and chronic periodontitis (CP) both characterize common chronic diseases that may have a reciprocal influence. Mechanisms such as vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition are observed in DM, which may contribute to the pathogenesis of periodontitis.[1]

Recently, Keles et al. observed the level and progression of YKL-40 in gingival crevicular fluid (GCF) and serum, among healthy, gingivitis, and CP individuals with or without DM, and it was found that the level was increased from gingivitis to periodontitis.[10] Kido et al. investigated the association between CP and DM with respect to the GCF YKL-40 level. They found that the level of YKL-40 was elevated in CP patients with or without type 2 DM (T2DM), but no increase was reported among healthy individuals.[2]

There is a paucity of literature regarding the effect of periodontal therapy on the GCF level of YKL-40. Hence, this clinico-biochemical study was conducted with intent to estimate the GCF level of YKL-40 in healthy controls and CP patients with and without DM at baseline and 6 weeks postperiodontal therapy (scaling and root planing [SRP]).

Research hypothesis (H1)

The GCF levels of YKL-40 APP are increased in periodontal diseases with and without type 2 diabetes, which decreases after periodontal therapy (SRP).

Null hypothesis (H0)

The GCF levels of YKL-40 APP do not increase in periodontal diseases with and without type 2 diabetes and do not decrease after periodontal therapy (SRP).


   Materials and Methods Top


The study approval was obtained from the Institutional Review Board and Ethical Committee (Ref. No. BDC/Exam/505/2014-15).

Estimation of sample size

Software G power version 3.1.9.2 was used.

Alpha: 0.05

Power of study: 0.8

Effect size: 0.4

Number of groups: 3

Estimated sample size per group will be 21.

Considering the number of dropouts and to increase the accuracy, we included 35 individuals in each group. Hence, our total study population consists of 105 individuals from the outpatient department of periodontics, with the age range of 30–60 years (mean age 42.69 ± 10.09 years). Written consent was obtained from all individuals. According to the classification by 1999 International World Workshop for periodontal diseases and conditions based on their clinical and radiographic features, they were divided into three equal groups, consisting of 35 individuals in each groups: healthy, CP with DM (glycated hemoglobin [HbA1c] 6%–7%), and CP without DM. Individuals were explained about the study both verbally and by written description which was signed by them before the commencement of the study.

Before the commencement of this study, the level of diabetes was analyzed by using HbA1c Now+ (A1 CNow+® system, Gujarat) to check whether the individuals are controlled diabetics (HbA1c 6.1%–7%). Proper history was taken to exclude the individuals who had taken antibiotics or any other drugs and any individuals who underwent periodontal treatment past 6 months. Furthermore, individuals with any systemic disease/conditions (apart from diabetes), tobacco habits, and smoking, individuals who were not willing to participate in the study, and pregnant and lactating mothers were excluded from the study.

Selection of individuals for each group was done after a brief and precise case history recording, as well as clinical and radiographic examination. The test site selection for GCF collection was based on the maximum scored sites, i.e., the site which shows the highest inflammatory signs (gingivitis) or greater amount of loss of attachment (CP), and stent was prepared for the same site. Clinical parameters such as plaque index (PI), gingival index (GI), probing pocket depth (PD), and clinical attachment level (CAL) were recorded at baseline and 6 weeks after SRP for Group II and Group III. The area of interest was gently cleared from the soft deposit, air-dried, and isolated with cotton rolls. 3 μL of GCF was collected from all three groups with the help of color coded 1–5 μL calibrated volumetric Hirschmann capillary pipette, which was instantly transferred to Eppendorf tube, and stored at –80°C till ELISA was performed. Both Group II and Group III were subjected to scaling on the same day and root planing past 1 week. Patients were called at 1 week interval to monitor oral hygiene status. After 6 weeks of SRP, patients in Group II and Group III were subjected to recording of clinical parameters followed by GCF collection.

Sandwich ELISA technique (RayBio®, Delhi) was performed for the quantitative measurement of YKL-40. The inner aspect of all the wells was precoated with a specific antibody for human YKL-40 (CHI3 L1). Both the samples and standards were added to the wells. Washing of the wells was repeated after addition of each component such as biotinylated anti-human YKL-40, horseradish peroxidase-conjugated streptavidin, and TMB (Tetramethyl benzidine) substrate solution. Finally, addition of stop solution changed the color from blue to yellow and the color intensity was measured at 450 nm.

The data analyses were made using the software IBM SPSS Statistics, Version 22 (Armonk, NY: IBM Corp). Kruskal–Wallis one-way analysis was performed among the three study groups for the comparison of clinical parameters scores and YKL-40 levels at baseline. Intergroup comparison was assessed by Mann–Whitney U-test. Baseline and posttherapy analyses of GCF YKL-40 in individual groups were performed by Wilcoxon signed-rank test.


   Results Top


The GCF samples were collected from all the participants. Sample collection was repeated again among 70 patients from Group II and Group III after phase 1 therapy. The intragroup comparison of clinical parameters (PI, GI, PD, and CAL) showed that its level decreased post-SRP [Table 1]. A significant reduction in the level of PI, GI, and PD was seen when inter- and intra-group comparison was done among Group I, Group II, and Group III [Table 1], which was statistically significant (P< 0.001). When the clinical parameters (PI, GI, PD, and CAL) among Group II and III were compared at baseline and 6 weeks posttherapy, it was observed that there was statistically significant (P< 0.001) reduction in the values [Table 1]. However, the same was not true during intergroup comparison between Group II and Group III, i.e., only GI score showed statistically significant change rest; all were not statistically significant. The level of GCF YKL-40 was compared at the baseline between Group I and Group II; furthermore, between Group I and Group III, it was observed that there was significant difference in the level of YKL-40. The mean GCF YKL-40 level after 6 weeks post-SRP significantly reduced for Group II and Group III as compared to their baseline [Figure 1].
Table 1: Inter- and intra-group comparison of clinical parameters at baseline and 6 weeks postscaling and root planing

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Figure 1: Inter- and intra-group comparison of gingival crevicular fluid YKL-40 among Group I (healthy individual), Group II (chronic periodontitis with diabetes mellitus), and Group III (chronic periodontitis)

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   Discussion Top


Periodontitis is an inflammatory condition of the oral cavity that ultimately leads to loss of tooth-supporting hard and soft tissues. Apart from the microorganisms and its toxic by-products, there are certain other mediators produced such as APPs, acute-phase reactants (APRs), cytokines, and prostaglandins. There are two main varieties of APPs: positive APPs and negative APPs. YKL-40 is a C-reactive protein which belongs to the positive APP variety.[2] It helps in regulating the vascular endothelial growth factor and has a role in inflammation, cell proliferation, differentiation, inflammation, remodeling of the extracellular matrix, and protection against apoptosis.[11] In CP, the inflammation is characterized by increase in the level of pro-inflammatory cytokines. Local production of cytokines leads to poor glycemic control in T2DM and increased level of pro-inflammatory biomarkers, thereby resulting in an exacerbation of ongoing inflammation.[12],[13] The presence of diabetes in a periodontitis patient may affect GCF levels of cytokines.

The study was undertaken to check the GCF level of YKL-40 in CP patients with and without T2DM, as well as to compare the clinical parameters in both Group II and Group III patients before and after SRP. GCF has been harvested from the gingival crevice or periodontal pocket using capillary micropipette tubes which is an atraumatic and noninvasive method.[14] We preferred ELISA test designed on the principle of “sandwich method” in our study because it offers a highly sensitive and specific method to analyze the contents of GCF. To assess the role of YKL-40 in the present study among periodontitis patients with and without diabetes, pre- and post-treatment records of PI and GI were undertaken. Apical shift of the junctional epithelium and loss of attachment are the hallmarks of periodontal diseases; therefore, the measurement of PD and CAL was also recorded using UNC-15 probe.

In the present study, the baseline mean plaque scores for Group II and Group III patients were significantly higher than in healthy group [Table 1]. Furthermore, on intergroup comparison, the difference in mean plaque score was statistically highly significant (P ≤ 0.001). Similar observations were made by Keles et al.,[11] who also reported higher plaque score in CP patients than in healthy groups. This finding validates the positive role played by the local factors in the etiopathogenesis of gingival and periodontal disease. The Group II and Group III patients underwent basic periodontal therapy (SRP) immediately after baseline recording of clinical parameters and collection of GCF. Rerecording of all clinical parameters was done after 6 weeks. It was found that the mean plaque scores were reduced in both the experimental groups, as compared to the baseline data [Table 1].

When the GI score for Group II and Group III was assessed at baseline, it was found that they are at higher level than Group I [Table 1]. On intergroup comparison, the difference in the mean GI score at the baseline was statistically highly significant (P ≤ 0.001). This may be explained by the fact that the amount of microbial plaque is directly related to the gingival status around the tooth. These findings are consistent with that of Kido et al.,[3] Keles et al.,[12] and George et al.[15] This reduction in plaque and gingival score as a result of SRP can be attributed to oral hygiene instructions given to the patients during the recall visits after SRP.[15],[16]

Intergroup comparison of periodontal PD values at baseline between Group II and Group III was higher than in Group I [Table 1]. This finding in our study is in agreement with that of Kido et al.[3] In the present study, CAL at baseline for Group II was reduced post SRP; similar findings were observed for Group III [Table 1]. The gain in CAL after SRP in the present study can be attributed to reduction in pathogenic bacteria and conversion of inflamed gingiva to healthy gingiva.[15],[16]

In this study, the GCF concentration of YKL-40 at baseline was found highest in CP (Group III) patients followed by CP-DM (Group II) and the least in Group I [Figure 1]. These results support the earlier finding by different studies in which it was reported that high mean CRP level among people with extensive periodontal destruction.[4],[17],[18],[19],[20] On the other hand, the level of YKL-40 in GCF was higher in patients with periodontal tissue breakdown, and the YKL-40 level slowly increased from healthy to gingivitis and then to periodontitis stage.[11] According to Kido et al.,[3] the level of GCF YKL-40 in CP patients with and without diabetes is greater than healthy controls. In our study, the baseline mean GCF YKL-40 level for Group II reduced at 6 weeks postperiodontal therapy follow-ups, which is aimed at eliminating the etiological factors for the disease component [Figure 1]. This observation was in accordance with the findings of the previous studies. On the contrary, the Group III (CP) patients also showed significant reduction in the GCF YKL-40 level when pre and posttreatment data were compared, which is in agreement with the other studies.[12],[21]

This study confirms the role of YKL-40 in periodontal inflammation. However, resolution of inflammation after periodontal therapy resulted in decreased GCF YKL-40 level. Another important observation made in this study was that the diabetic status does not seem to influence the YKL-40 level in CP if the HbA1c is under control. The final outcomes of this study deserve further considerations in the development of preventive and therapeutic methods for treating periodontal disease. However, additional studies involving larger sample size, with longer follow-ups, are required to affirm these results and also to better understand the role of YKL-40 in periodontal health and disease.


   Conclusion Top


Within the limitations of our study, we can conclude that the level of YKL-40 is significantly higher in both CP with and without DM compared to the healthy individuals. Hence, we conclude that basic periodontal therapy (SRP) in both the diseased group leads to significant reduction in GCF level of YKL-40, thereby helping to improve the periodontal status of the individual. This study also reaffirms that YKL-40 can be used as a diagnostic biomarker in periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Grossi SG, Genco RJ, Machtei EE, Ho AW, Koch G, Dunford R, et al. Assessment of risk for periodontal disease. II. Risk indicators for alveolar bone loss. J Periodontol 1995;66:23-9.  Back to cited text no. 1
    
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Polepalle T, Moogala S, Boggarapu S, Pesala DS, Palagi FB. Acute phase proteins and their role in periodontitis: A Review. J Clin Diagn Res 2015;9:ZE01-5.  Back to cited text no. 2
    
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Kido J, Bando Y, Bando M, Kajiura Y, Hiroshima Y, Inagaki Y, et al. YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes. Oral Dis 2015;21:667-73.  Back to cited text no. 3
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