Effects of scaling and root planing on gingival crevicular fluid vascular endothelial growth factor level in chronic periodontitis patients with and without diabetes mellitus: A clinicobiochemical study.

AIM: To estimate the gingival crevicular fluid (GCF) level of vascular endothelial growth factor (VEGF) in periodontally healthy controls and chronic periodontitis (CP) patients with and without diabetes mellitus (DM) and also to investigate the effect of scaling and root planing (SRP) on the GCF VEGF level.
MATERIALS AND METHODS: One hundred and five patients were divided into three groups: Healthy (Group 1), CP (Group 2), and CP with DM (Group 3). Group 2 and Group 3 patients underwent SRP planning, and the cases were followed for 6 weeks. Periodontal clinical parameters such as plaque index, gingival index, probing pocket depth, and clinical attachment level were recorded at baseline and 6-week posttherapy. GCF samples collected from each patient were quantified for VEGF level using enzyme-linked immunosorbent assay.
RESULTS: The mean GCF VEGF level was increased in CP patients with and without DM compared to healthy patients and SRP therapy caused a statistically significant (P < 0.001) reduction in GCF VEGF level.
CONCLUSION: VEGF is increased in GCF of CP patients with and without DM and that SRP substantially reduces its level in GCF.

INTRODUCTION

Chronic periodontitis (CP) is the infection of highly vascularizing supporting periodontal tissues with episodes of active destruction and periods of quiescence. Earlier reports indicated that in the region of the periodontal pocket, there was a positive relationship between an increase in the number of blood vessels and progression of the disease, especially affecting capillaries and venules.[123]

Angiogenesis (neovascularization) is defined as the process of budding of new capillaries and formation of new blood vessels, considered to be the integral component in the development of chronic inflammatory disease. Angiogenesis can increase the severity of the inflammation by virtue of the new blood vessels that can transport pro-inflammatory cells and supply nutrients and oxygen to the inflamed tissues. Of the various cytokines and growth factors that are involved in the regulation of angiogenesis, the most potent agent that acts specifically on vascular endothelium is vascular endothelial growth factor (VEGF).[14]

VEGF is a multifunctional angiogenic cytokine of importance and an endothelial-specific growth factor that potently increases microvascular permeability, stimulates endothelial cell (EC) proliferation, and induces proteolytic enzyme expression and the migration of ECs, monocytes, and osteoblasts, all of which are essential for angiogenesis.[5] VEGF shares homology with platelet-derived growth factor and placenta growth factor (PlGF) and has recently attracted attention as a potential inducer of vascular permeability and angiogenesis. Several members of the VEGF family have been described during the past few years, including VEGF-A, PlGF, VEGF-B, VEGF-C, VEGF-D, and VEGF-E.[67] VEGF has been detected in human periodontal tissues and gingival crevicular fluid (GCF) in variable quantity in healthy and diseased sites. In periodontal tissues, VEGF is detectable within EC, plasma cells, macrophages and in junctional, sulcular, and gingival epithelium.[8]

Diabetes mellitus (DM) is a metabolic disease usually characterized by the classic triad of polydipsia, polyuria, and polyphagia, the consequences of homeostasis disruption, due to impaired glucose metabolism.[9] Periodontal disease has been proposed as the sixth complication of diabetes, based on the frequent presence of both diseases in the same patient.[1011] Mechanisms such as vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition, which are observed in DM, may contribute to periodontitis.[12] VEGF not only appears to play a central role in mediating diabetic vasculopathy in many organs but also affects glucose levels, and ultimately, the extent of diabetic complications.[6]

Thus, this study aimed at estimating the GCF levels of VEGF in healthy controls and CP patients with and without DM and also to evaluate the effect of periodontal therapy (scaling and root planing [SRP]) in these patients. The null hypothesis stated that there was no difference in GCF levels of VEGF in healthy controls and CP patients with and without DM, and Phase-I periodontal therapy does not affect these levels in CP patients with and without DM. The research hypothesis stated that there was a difference in GCF levels of VEGF in healthy controls and CP patients with and without DM, and Phase-I periodontal therapy does affect these levels in CP patients with and without DM.

MATERIALS AND METHODS

Study population

For this study, 105 patients (35 systemically healthy, 35 CP, and 35 CP patients with Type II DM) in the age range of 30–60 years (mean age 41.3 ± 7.8 years) were recruited from the outpatient Department of Periodontics, from May 2013 to November 2013 Table 1. The study was approved by the Institutional Review Board, and all study patients were given a detailed verbal and written description of the study and signed an informed consent form before commencement of the study.

Table 1

Demographic variables

The diagnosis of healthy and CP patients was made on the basis of clinical and radiographic criteria proposed by the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions.[13] The most severely affected tooth was used as test site for the evaluation of the clinical parameters and GCF sampling. All the selected 105 patients were divided into three groups of 35 each: Healthy subject (Group 1), CP patients without DM (Group 2), and CP with well-controlled type II DM (HbA1c level 6–8%) (Group 3).[14] During the study, no change in the medication regime of the diabetic patients was performed. Patients with any history of a systemic disease, those having taken any antibiotic therapy or underwent any form of periodontal treatment in the past 6 months, pregnant or lactating females, smokers, and those on antiviral or immunosuppressive drugs, were excluded from the study.

Clinical examination

All the measurements were performed by single precalibrated examiner. At baseline, all the selected cases were subjected to recording of clinical parameters such as plaque index (PI),[15] gingival index (GI),[16] probing pocket depth (PD), and clinical attachment level (CAL).[17] Probing PD was performed at six sites per tooth. For Group 2 and Group 3 patients, clinical parameters were also recorded at 6 weeks after SRP. HbA1c levels of diabetic patients were assessed by using chairside test kit (Bayer A1CNow+®, Ontario, Canada).

Site selection and collection of gingival crevicular fluid

After a brief and precise case history recording, the site showing the greatest attachment loss was selected for GCF sample collection in Groups 2 and 3. In the healthy group, one of the maxillary teeth was selected for GCF sample collection. The selected site was air dried and isolated with cotton rolls. Supragingival plaque was removed gently without touching the marginal gingiva, to avoid contamination and blocking of the microcapillary pipette.

GCF was collected by placing white color-coded 1–5 µL calibrated volumetric microcapillary pipettes (Sigma-Aldrich, St. Louis, USA). By using extra-crevicular (unstimulated) method, a standardized volume of 1 µL GCF was collected from each test site. The test sites, which did not express standard volume (1 µL) of GCF, or micropipette contaminated with blood/saliva/plaque were excluded or discarded. Samples of GCF were collected at the initial visit in Group 1, Group 2, and Group 3 patients. SRP was performed for Group 2 and Group 3 patients at the same appointment after GCF collection. SRP was done using ultrasonic scalers and area specific Gracey curettes (Osung, Gyeonggi, South Korea). After 6 weeks, GCF was collected from same site of the patients in Group 2 and Group 3. Collected samples were immediately transferred to plastic tubes and stored at − 80°C until the time of the assay. For this 6-week study, patients were called at 1-week interval to monitor oral hygiene status.

Vascular endothelial growth factor assay

The concentration of VEGF was determined by quantitative sandwich enzyme-linked immunosorbent assay kit (Krishgen Biosystems, Mumbai, India). This assay employs an antibody specific for human VEGF coated on a 96-well plate. Standards and samples were pipetted into the wells, and VEGF present in a sample was bound to the wells by the immobilized antibody. The wells were washed and biotin-conjugated anti-human VEGF antibody is added. After washing away unbound biotin-conjugated antibody, horseradish peroxidase-conjugated streptavidin was pipetted to the wells. The wells were again washed, 3,3',55'- tetramethylbenzidine substrate solution was added to the wells and color developed in proportion to the amount of VEGF bound. The stop solution changed the color from blue to yellow, and the intensity of the color was measured at 450 nm. The concentrations of VEGF in the tested samples were estimated using the standard curve plotted using the optical density values with the standards.

Statistical analyses

Data were analyzed using a software program (SPSS Statistical Package [PC version 19.0], SPSS, Chicago, IL, USA). Kruskal–Wallis test was performed to compare clinical parameters of the three groups at baseline. ANOVA Tukey's test was performed to compare GCF VEGF level of the three groups at baseline. Wilcoxon's signed rank test was performed to determine whether there was any difference between GCF VEGF level before and after treatment. Mann–Whitney U test was used for intergroup comparison.

RESULTS

A total of 175 samples were collected (105 samples at baseline and 70 samples 6 weeks postperiodontal therapy from Groups 2 and 3) from 105 volunteers. There were no dropouts during the study. All the samples collected tested positive for the presence of VEGF. A significant (P < 0.001) reduction in PI, GI, probing PD, and CAL in both Group 2 and Group 3 occurred at 6 weeks after Phase-I treatment, compared with baseline [Table 2]. However, when the clinical parameters were compared between Group 2 and Group 3 at baseline and at 6 weeks after Phase-I therapy, no statistically significant change was seen [Table 3].

Table 2

Comparative evaluation of clinical parameters at baseline and 6 weeks after periodontal treatment

Table 3

Intergroup comparison of clinical parameters and gingival crevicular fluid vascular endothelial growth factor levels at baseline and 6 weeks after Phase-I therapy

Levels of vascular endothelial growth factor in gingival crevicular fluid

The mean GCF VEGF levels in all the three groups are presented in Table 2. At baseline, there was a significant difference in GCF VEGF levels between Groups 1 and 2 and Groups 1 and 3 (P < 0.001). Furthermore, there was a statistically highly significant difference in the GCF VEGF level at baseline and 6 weeks posttherapy in Groups 2 and 3 (P < 0.001) [Table 2]. When the mean GCF VEGF levels of Groups 2 and 3 were compared at baseline, no statistically significant changes were seen. However, when the levels were compared 6 weeks after Phase-I therapy, a statistically significant change was seen [Table 3].

DISCUSSION

Periodontitis is a chronic inflammatory disease with episodes of active destruction and periods of quiescence. However, patients vary in their response to chronic gingival inflammation. Some are highly susceptible to rapidly destructive disease and others, who have a similar extent of periodontal inflammation, are more resistant to destructive disease.[1819]

The periodontal vasculature is profoundly affected during progression of periodontal disease. Angiogenesis refers to a process by which new blood vessels are produced by sprouting from established vessels. Angiogenesis can contribute to the severity of the inflammation as a result of the ability of new blood vessels to transport pro-inflammatory cells to the lesion and supply nutrients and oxygen to the inflamed tissue.[8] Numerous cytokines and growth factors are involved in the regulation of angiogenesis; however, most potent agent that act specifically on vascular endothelium is VEGF.[2021] Periodontitis which is an inflammatory response to a bacterial challenge represents a portal of entry for periodontal pathogens, bacterial endotoxins, and pro-inflammatory cytokines. Thus, the local oral inflammatory disease, periodontitis, may induce and perpetuate a systemic inflammation that may aggravate systemic diseases such as cardiovascular disease, pulmonary disease, rheumatoid arthritis, and DM.[22] Periodontitis is frequently mentioned among the oral problems observed in DM, which may contribute to periodontitis by mechanisms such as vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition. VEGF is one of the major factors promoting diabetic complications and is implicated in the development of neovascularization and endothelial dysfunction in diabetic vascular complications.[2324]

Considering the important role of VEGF in the pathogenesis of CP, it is important to control the VEGF level in the local inflammatory lesion by periodontal treatment to inhibit the disease progression successfully in periodontitis model. Studies have shown that SRP is an effective means of slowing or arresting the periodontal disease progression and severity.[2526]

VEGF is a multifunctional cytokine that plays a pivotal role during inflammation by mediating neovascularization. Studies have shown that the volume of GCF and total amount of VEGF were greater in diseased sites compared to clinically healthy sites.[18] Contrary to this finding, Booth et al.[18] reported a higher concentration of VEGF in GCF of healthy patients than periodontally diseased sites, and explained their observation with two possible mechanisms:First, the presence of subclinical levels of inflammation or healing after the microbial assault that may interfere with VEGF expression, and second, physiological angiogenesis in the gingival/periodontal environment. It was further stated that high individual variance may also affect the overall results. However, our results did not match this finding probably due to differences in patient selection criteria and comparison methods.

In this study, the difference in the mean GCF concentration of VEGF of Group 2 and Group 3 was statistically highly significant compared to Group 1. This is in agreement with the findings presented in the study conducted by Mohamed et al.[27] However, there was no statistically significant difference in the mean GCF concentration of VEGF between Group 2 and Group 3 even though the clinical parameters recorded in Group 3 at baseline was comparable to that recorded for Group 2. Therefore, it can be interpreted that the presence of well-controlled diabetes does not have an effect on the expression of VEGF in GCF of CP patients and that it is the clinical parameters that would dictate its expression. These findings are consistent with the observations made by Güneri et al.,[1] Prapulla et al.,[5] and Booth et al.[18] In addition, Sakallioglu et al. in his recent study stated that the level of VEGF may increase in periodontitis patients which is in agreement with the findings of our study.[28]

There was a statistically highly significant reduction in the mean GCF VEGF level 6 weeks after Phase-I periodontal therapy (SRP) in Groups 2 and 3. This finding is consistent with the results published by Padma et al. in their study.[29] The reduction in the GCF VEGF level after Phase-I periodontal therapy can be attributed to the resolution of gingival inflammation and reduction in PD due to tissue shrinkage. Moreover, when the GCF VEGF levels of Groups 2 and 3 were compared 6 weeks after Phase-I therapy, a statistically significant difference was seen. This finding can be interpreted as that, even though the presence of diabetes in CP patients may not have an effect on expression of VEGF in GCF, the effectiveness of Phase-I therapy in controlling VEGF levels is poorer in patients with both diabetes and CP. Taken together, these results indicate that GCF VEGF expression is higher in periodontitis patients than in healthy patients, which is in agreement with the studies done by Güneri et al.[1] and Prapulla et al.[5] where they reported progressive increase in GCF mean concentrations of VEGF from healthy to periodontitis patients. The variability of GCF VEGF levels within the CP group can be attributed to the different stages of disease process at the time of GCF collection. Further, the presence of well-controlled DM in CP patients did not influence the level of VEGF in GCF. Furthermore, the treatment aimed at arresting periodontal disease progression resulted in statistically significant reduction in the levels of VEGF in GCF. The limitations of this study however that is, a limited sample size was included. In addition, the VEGF analysis was nonspecific. A specific analysis will further throw light on the role VEGF may play in the periodontal pathophysiology. Further, longitudinal prospective studies involving larger sample population are needed to confirm these findings and to better understand the role of VEGF in periodontal health and disease.

CONCLUSION

Taking all these into consideration, GCF VEGF can be considered a potential biomarker for periodontal disease and deserves further considerations in the development of preventive and therapeutic methods for treating periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.