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 . 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 ) . 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 . 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 . 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 . 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 . periodontal disease has been proposed as the sixth complication of diabetes , based on the frequent presence of both diseases in the same patient . mechanisms such as vascular changes , neutrophil dysfunction , altered collagen synthesis , and genetic predisposition , which are observed in dm , may contribute to periodontitis . 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 . 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 . for this study , 105 patients ( 35 systemically healthy , 35 cp , and 35 cp patients with type ii dm ) in the age range of 3060 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 . 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 . 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 68% ) ( group 3 ) . during the study , 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 . all the selected cases were subjected to recording of clinical parameters such as plaque index ( pi ) , gingival index ( gi ) , probing pocket depth ( pd ) , and clinical attachment level ( cal ) . probing pd was performed at six sites per tooth . for group 2 and group 3 patients , hba1c levels of diabetic patients were assessed by using chairside test kit ( bayer a1cnow+ , ontario , canada ) . 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 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 15 l calibrated volumetric microcapillary pipettes ( sigma - aldrich , st . the test sites , which did not express standard volume ( 1 l ) 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  80c until the time of the assay . for this 6-week study , patients were called at 1-week interval to monitor oral hygiene status . the concentration of vegf was determined by quantitative sandwich enzyme - linked immunosorbent assay kit ( krishgen biosystems , mumbai , india ) . 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 . data were analyzed using a software program ( spss statistical package [ pc version 19.0 ] , spss , chicago , il , usa ) . 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 . for this study , 105 patients ( 35 systemically healthy , 35 cp , and 35 cp patients with type ii dm ) in the age range of 3060 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 . 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 . 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 68% ) ( group 3 ) . during the study , 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 . all the selected cases were subjected to recording of clinical parameters such as plaque index ( pi ) , gingival index ( gi ) , probing pocket depth ( pd ) , and clinical attachment level ( cal ) . probing pd was performed at six sites per tooth . for group 2 and group 3 patients , hba1c levels of diabetic patients were assessed by using chairside test kit ( bayer a1cnow+ , ontario , canada ) . 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 15 l calibrated volumetric microcapillary pipettes ( sigma - aldrich , st . the test sites , which did not express standard volume ( 1 l ) 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  80c until the time of the assay . for this 6-week study , patients were called at 1-week interval to monitor oral hygiene status . the concentration of vegf was determined by quantitative sandwich enzyme - linked immunosorbent assay kit ( krishgen biosystems , mumbai , india ) . 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 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 . data were analyzed using a software program ( spss statistical package [ pc version 19.0 ] , spss , chicago , il , usa ) . 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 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 . 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 ] . comparative evaluation of clinical parameters at baseline and 6 weeks after periodontal treatment intergroup comparison of clinical parameters and gingival crevicular fluid vascular endothelial growth factor levels at baseline and 6 weeks after phase - i therapy 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 ] . 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 ] . 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 . 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 . numerous cytokines and growth factors are involved in the regulation of angiogenesis ; however , most potent agent that act specifically on vascular endothelium is vegf . 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 . 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 . 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 . 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 . 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 . 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 . 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 gneri et al . , prapulla et al . , and booth et al . 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 . 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 . 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 gneri et al . and prapulla et al . 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 . 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 . 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 .
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 .