Short term effects of non surgical perio
archives of oral biology 58 (2013) 391–396
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Short term effects of non-surgical periodontal treatment
on gingival crevicular fluid levels of tissue plasminogen
activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2)
in patients with chronic and aggressive periodontitis§
Gülay Tüter a,*, Burcu Özdemir a, Bülent Kurtiş a, Muhittin Serdar b,
Ayşegül Atak Yücel c, Eylem Ayhan a
a
Gazi University, Faculty of Dentistry, Department of Periodontology, Ankara, Turkey
Gülhane Military Medical Academy, Department of Biochemistry, Ankara, Turkey
c
Gazi University, Faculty of Medicine, Department of Immunology, Ankara, Turkey
b
article info
abstract
Article history:
Objective: The purpose of this study was to evaluate the gingival crevicular fluid (GCF) levels
Accepted 10 August 2012
of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) in
Keywords:
subjects, before (BT) and after (AT) the non-surgical periodontal treatment.
Tissue plasminogen activator
Design: Systemically healthy 12 CP and 13 AgP patients and 20 control subjects were included
aggressive periodontitis (AgP), chronic periodontitis (CP) and periodontally healthy control
Plasminogen activator inhibitor 2
in this study. Plaque index, gingival index, probing depth and clinical attachment levels were
Periodontal diseases
recorded and GCF samples were collected BT and AT. Assays for GCF t-PA and PAI-2 levels were
Gingival crevicular fluid
carried out by an enzyme linked immunosorbent assay (ELISA). The x2, Wilcoxon and Mann–
Whitney U tests and Spearman correlation coefficient were used for data analyses.
Results: Statistically significant reductions in clinical index scores were noted in both periodontitis groups after treatment. No significant differences were detected in GCF levels of t-PA
and PAI-2 between CP and AgP groups at either BT or AT. There was a statistically significant
decrease in GCF PAI-2 levels in CP after therapy ( p < 0.01). GCF t-PA levels in CP and AgP groups
exhibited significant correlations with PD and CAL measurements at both BT and AT ( p < 0.01).
Conclusion: Significant decrease was detected for GCF PAI-2 levels in CP and clinical parameters in both CP and AgP by non-surgical periodontal treatment.
# 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Plasminogen activator system plays a crucial role in many
physiological processes, including tissue remodelling, cell
migration, wound healing angiogenesis, as well as pathological events such as acute and chronic inflammatory reactions,
tumour invasion and metastasis.1 Proenzyme plasminogen is
§
converted to active plasmin by plasminogen activator system
which is regulated by protease activators, tissue type
plasminogen activator (t-PA) and urokinase type plasminogen
activator (u-PA) and their inhibitors, plasminogen activator
inhibitors 1 (PAI-1) and 2 (PAI-2).2
Plasmins act as proinflammatory agents as they induce
neutrophil aggregation, platelet degranulation, syntheses and
secretion of arachidonic acid derivates and stimulate the
GCF t-PA and PAI-2 levels may be used as markers to assess periodontal disease and treatment efficiency.
* Corresponding author at: Department of Periodontology, Faculty of Dentistry, Gazi University, Biskek Caddesi 82, Sokak 06510 Emek,
Ankara, Turkey. Tel.: +90 532 2262425.
E-mail address: gulay@gazi.edu.tr (G. Tüter).
0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2012.08.008
392
archives of oral biology 58 (2013) 391–396
release of inflammatory cytokines tumour necrosis factoralpha (TNF-a), interleukin (IL)-1-alpha (IL-1-a) and -beta (IL1b).1,3–5 They directly degrade extracellular matrix (ECM) or
contribute to their destruction via activation of matrix
metalloproteinases (MMPs) and blocking the effects of tissue
inhibitors of MMPs.6 The proteolytic activity of plasminogen
activators are inhibited by PAI-1 which is primarily produced
by endothelial and malignant cells, and PAI-2 which is mainly
produced by monocytes, macrophages, epithelial cells and
fibroblasts.7,8
Periodontal diseases have long known to have complex
pathogenesis and they have been proposed that it is the host
response to the long-term bacterial challenge.9 The periodontal connective tissue ECM provides the environment for all cell
interactions occurring during development, pathology, and
regeneration processes.10 During periodontitis, these structures and components are significantly disrupted and periodontitis is characterized by the loss of collagen fibres and
other ECM constituents of tooth supporting periodontal
tissues. A possible mechanism for the degradation of
periodontal ECM is the independent and/or cooperative action
of both human and bacterial proteinases.11
It is well known that the least invasive approach to
examine destructive process of periodontal disease involves
analysis of gingival crevicular fluid (GCF), an inflammatory
exudate present in the gingival crevice.12 Previously, t-PA, uPA, PAI-1 and PAI-2 were detected in GCF.13 Furthermore, high
concentrations of t-PA and PAI-2 in GCF were indicated and it
was suggested that they were locally produced and might be
involved in the aggravation of gingival inflammation.14 Yin
et al. stated that t-PA and PAI-2 might play a significant role in
the periodontal tissue destruction and tissue remodelling,
and that t-PA and PAI-2 in GCF might be used as clinical
markers to evaluate the periodontal diseases and assess
treatment.15 Previously some studies13,15,16 examined t-PA
and PAI-2 levels in GCF of healthy subjects, periodontitis and
gingivitis patients and observed the alterations after the
periodontal treatment. However there is still lack of information on GCF t-PA and PAI-2 levels of different forms of
periodontitis. We believe that evaluation of t-PA and PAI-2
levels in chronic and aggressive periodontitis will contribute
to better understanding of periodontal disease pathogenesis
and periodontal treatment.
The aims of the present study were to evaluate the effect of
non-surgical periodontal treatment on clinical parameters
and to compare GCF levels of t-PA and PAI-2 in chronic and
aggressive periodontitis patients, in addition to investigate the
relationship between clinical parameters and GCF t-PA and
PAI-2 levels.
2.
Materials and methods
2.1.
Study population and study design
A total of 12 chronic periodontitis (CP) (8 males and 4 females
with a mean age of 44 8 years), 13 aggressive periodontitis
(AgP) (5 males and 8 females with a mean age of 26 4 years)
and 20 clinically healthy control group subjects (C) (7 males
and 13 females with a mean age of 29 3 years) were included
in the present study. Control group subjects had no history of
periodontal disease and all participants were recruited from
individuals referred to Gazi University, Faculty of Dentistry,
Department of Periodontology either for dental treatment or
only for dental check-up, between March 2008 and December
2009. The protocol of this study was approved by the Ethical
Committee of the Faculty of Dentistry, Gazi University,
Ankara, Turkey and the study was conducted in accordance
with the Helsinki Declaration of 1975, as revised in 2000.
Informed written consent was obtained from all subjects after
the details of the clinical procedures, including periodontal
measurements and GCF sampling were explained.
Periodontal disease status was determined according to
clinical and radiographic criteria. The classifications of the
study groups were made according to criteria proposed by the
1999 International World Workshop for a Classification of
Periodontal Disease and Conditions.17 Chronic periodontitis
patients showing radiographic evidence of bone loss and
attachment loss with a minimum of 8 teeth having periodontal
pockets greater than 4 mm were included into the study.
Diagnosis of aggressive periodontitis was determined according
to the following criteria: patients 6 mm and with
radiographic evidence of alveolar bone loss were taken into
consideration. At least three of these eight teeth with probing
depth >6 mm were not first molars or incisors. Other factors
such as family aggregation, rapid progression and the relationship between local factors and periodontal destruction were
also considered. Rapid progression was determined according
to clinical, radiographic and etiologic manifestations of aggressive periodontitis. All subjects were in good general health, were
non-smokers and none had received periodontal treatment
during the past 6 months or received antibiotic medication
during the past 3 months. No subjects with a history of systemic
conditions such as heart disease, diabetes and other types of
disorders which could influence the course of periodontal
disease were enrolled. Enrolled subjects did not use a medication that could affect the manifestations of periodontal disease,
such as antibiotics, phenytoin, cyclosporine, anti-inflammatory
drugs, or calcium channel blockers. None of the women were
pregnant in postmenopausal or lactation period.
Periodontal clinical measurements and non-surgical
2.2.
periodontal therapy
Inter and intra-examiner calibrations were performed prior to
initiation of the study. Inter-examiner reliability was determined by having each examiner make dual measurements
along with those of the Project Director on at least 2 patients,
and to remediate any measurement differences >1 mm or 1
score. Intra-examiner reliability was determined by taking
repeated measurements from the same pocket at the calibration session and on about 20% of the cases (randomly
determined) during the study.
Prior to any treatment, baseline clinical indices were
recorded, and then GCF samples were collected. The clinical
recordings included plaque index (PI),18 gingival index (GI),19
probing depth (PD), and clinical attachment level (CAL).
Clinical measurements performed on six sites per
tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual,
archives of oral biology 58 (2013) 391–396
mid-lingual, disto-lingual) using with a Williams periodontal
probe (Nordent Manufacturing Inc., Elk Grove Vilage, IL, USA)
calibrated in millimetres. The deepest six pockets of each
subject were chosen for clinical recordings and GCF sampling.
Therefore, for obtaining GCF samples, 72 (12 CP patients 6
sites) pockets were sampled in CP group, 78 (13 AgP
patients 6 sites) pockets were sampled in AgP group and
120 (20 participants 6) pockets were sampled in C group at
each of the test appointments.
Periodontitis patients received non-surgical periodontal
treatment consisted of oral hygiene instruction, scaling and
root planing with ultrasonic and hand instruments, which was
performed without any adjunctive therapy and completed
within 2 weeks from the beginning of the study. After 6 weeks
from baseline measurements, clinical measurements and GCF
sampling were repeated for periodontitis groups.
2.3.
analysis among the groups, One-Sample Kolmogorow–Simirnov test was performed. According to the results of this test,
non-parametric analysis was determined. The results were
shown as median and 25–75 percentiles in the parentheses
(quarters). The x2 test was used to determine whether there
was a statistical difference among the groups with respect to
gender. Wilcoxon test was used for evaluation of the
differences before and after the treatment. Mann–Whitney
U test was used for evaluation of the differences between CP
and C and also between AgP and C before and after the
treatment. The correlations between clinical indices and GCF
t-PA and PAI-2 levels were analyzed with Spearman correlation coefficient. p-Value
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Short term effects of non-surgical periodontal treatment
on gingival crevicular fluid levels of tissue plasminogen
activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2)
in patients with chronic and aggressive periodontitis§
Gülay Tüter a,*, Burcu Özdemir a, Bülent Kurtiş a, Muhittin Serdar b,
Ayşegül Atak Yücel c, Eylem Ayhan a
a
Gazi University, Faculty of Dentistry, Department of Periodontology, Ankara, Turkey
Gülhane Military Medical Academy, Department of Biochemistry, Ankara, Turkey
c
Gazi University, Faculty of Medicine, Department of Immunology, Ankara, Turkey
b
article info
abstract
Article history:
Objective: The purpose of this study was to evaluate the gingival crevicular fluid (GCF) levels
Accepted 10 August 2012
of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) in
Keywords:
subjects, before (BT) and after (AT) the non-surgical periodontal treatment.
Tissue plasminogen activator
Design: Systemically healthy 12 CP and 13 AgP patients and 20 control subjects were included
aggressive periodontitis (AgP), chronic periodontitis (CP) and periodontally healthy control
Plasminogen activator inhibitor 2
in this study. Plaque index, gingival index, probing depth and clinical attachment levels were
Periodontal diseases
recorded and GCF samples were collected BT and AT. Assays for GCF t-PA and PAI-2 levels were
Gingival crevicular fluid
carried out by an enzyme linked immunosorbent assay (ELISA). The x2, Wilcoxon and Mann–
Whitney U tests and Spearman correlation coefficient were used for data analyses.
Results: Statistically significant reductions in clinical index scores were noted in both periodontitis groups after treatment. No significant differences were detected in GCF levels of t-PA
and PAI-2 between CP and AgP groups at either BT or AT. There was a statistically significant
decrease in GCF PAI-2 levels in CP after therapy ( p < 0.01). GCF t-PA levels in CP and AgP groups
exhibited significant correlations with PD and CAL measurements at both BT and AT ( p < 0.01).
Conclusion: Significant decrease was detected for GCF PAI-2 levels in CP and clinical parameters in both CP and AgP by non-surgical periodontal treatment.
# 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Plasminogen activator system plays a crucial role in many
physiological processes, including tissue remodelling, cell
migration, wound healing angiogenesis, as well as pathological events such as acute and chronic inflammatory reactions,
tumour invasion and metastasis.1 Proenzyme plasminogen is
§
converted to active plasmin by plasminogen activator system
which is regulated by protease activators, tissue type
plasminogen activator (t-PA) and urokinase type plasminogen
activator (u-PA) and their inhibitors, plasminogen activator
inhibitors 1 (PAI-1) and 2 (PAI-2).2
Plasmins act as proinflammatory agents as they induce
neutrophil aggregation, platelet degranulation, syntheses and
secretion of arachidonic acid derivates and stimulate the
GCF t-PA and PAI-2 levels may be used as markers to assess periodontal disease and treatment efficiency.
* Corresponding author at: Department of Periodontology, Faculty of Dentistry, Gazi University, Biskek Caddesi 82, Sokak 06510 Emek,
Ankara, Turkey. Tel.: +90 532 2262425.
E-mail address: gulay@gazi.edu.tr (G. Tüter).
0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2012.08.008
392
archives of oral biology 58 (2013) 391–396
release of inflammatory cytokines tumour necrosis factoralpha (TNF-a), interleukin (IL)-1-alpha (IL-1-a) and -beta (IL1b).1,3–5 They directly degrade extracellular matrix (ECM) or
contribute to their destruction via activation of matrix
metalloproteinases (MMPs) and blocking the effects of tissue
inhibitors of MMPs.6 The proteolytic activity of plasminogen
activators are inhibited by PAI-1 which is primarily produced
by endothelial and malignant cells, and PAI-2 which is mainly
produced by monocytes, macrophages, epithelial cells and
fibroblasts.7,8
Periodontal diseases have long known to have complex
pathogenesis and they have been proposed that it is the host
response to the long-term bacterial challenge.9 The periodontal connective tissue ECM provides the environment for all cell
interactions occurring during development, pathology, and
regeneration processes.10 During periodontitis, these structures and components are significantly disrupted and periodontitis is characterized by the loss of collagen fibres and
other ECM constituents of tooth supporting periodontal
tissues. A possible mechanism for the degradation of
periodontal ECM is the independent and/or cooperative action
of both human and bacterial proteinases.11
It is well known that the least invasive approach to
examine destructive process of periodontal disease involves
analysis of gingival crevicular fluid (GCF), an inflammatory
exudate present in the gingival crevice.12 Previously, t-PA, uPA, PAI-1 and PAI-2 were detected in GCF.13 Furthermore, high
concentrations of t-PA and PAI-2 in GCF were indicated and it
was suggested that they were locally produced and might be
involved in the aggravation of gingival inflammation.14 Yin
et al. stated that t-PA and PAI-2 might play a significant role in
the periodontal tissue destruction and tissue remodelling,
and that t-PA and PAI-2 in GCF might be used as clinical
markers to evaluate the periodontal diseases and assess
treatment.15 Previously some studies13,15,16 examined t-PA
and PAI-2 levels in GCF of healthy subjects, periodontitis and
gingivitis patients and observed the alterations after the
periodontal treatment. However there is still lack of information on GCF t-PA and PAI-2 levels of different forms of
periodontitis. We believe that evaluation of t-PA and PAI-2
levels in chronic and aggressive periodontitis will contribute
to better understanding of periodontal disease pathogenesis
and periodontal treatment.
The aims of the present study were to evaluate the effect of
non-surgical periodontal treatment on clinical parameters
and to compare GCF levels of t-PA and PAI-2 in chronic and
aggressive periodontitis patients, in addition to investigate the
relationship between clinical parameters and GCF t-PA and
PAI-2 levels.
2.
Materials and methods
2.1.
Study population and study design
A total of 12 chronic periodontitis (CP) (8 males and 4 females
with a mean age of 44 8 years), 13 aggressive periodontitis
(AgP) (5 males and 8 females with a mean age of 26 4 years)
and 20 clinically healthy control group subjects (C) (7 males
and 13 females with a mean age of 29 3 years) were included
in the present study. Control group subjects had no history of
periodontal disease and all participants were recruited from
individuals referred to Gazi University, Faculty of Dentistry,
Department of Periodontology either for dental treatment or
only for dental check-up, between March 2008 and December
2009. The protocol of this study was approved by the Ethical
Committee of the Faculty of Dentistry, Gazi University,
Ankara, Turkey and the study was conducted in accordance
with the Helsinki Declaration of 1975, as revised in 2000.
Informed written consent was obtained from all subjects after
the details of the clinical procedures, including periodontal
measurements and GCF sampling were explained.
Periodontal disease status was determined according to
clinical and radiographic criteria. The classifications of the
study groups were made according to criteria proposed by the
1999 International World Workshop for a Classification of
Periodontal Disease and Conditions.17 Chronic periodontitis
patients showing radiographic evidence of bone loss and
attachment loss with a minimum of 8 teeth having periodontal
pockets greater than 4 mm were included into the study.
Diagnosis of aggressive periodontitis was determined according
to the following criteria: patients 6 mm and with
radiographic evidence of alveolar bone loss were taken into
consideration. At least three of these eight teeth with probing
depth >6 mm were not first molars or incisors. Other factors
such as family aggregation, rapid progression and the relationship between local factors and periodontal destruction were
also considered. Rapid progression was determined according
to clinical, radiographic and etiologic manifestations of aggressive periodontitis. All subjects were in good general health, were
non-smokers and none had received periodontal treatment
during the past 6 months or received antibiotic medication
during the past 3 months. No subjects with a history of systemic
conditions such as heart disease, diabetes and other types of
disorders which could influence the course of periodontal
disease were enrolled. Enrolled subjects did not use a medication that could affect the manifestations of periodontal disease,
such as antibiotics, phenytoin, cyclosporine, anti-inflammatory
drugs, or calcium channel blockers. None of the women were
pregnant in postmenopausal or lactation period.
Periodontal clinical measurements and non-surgical
2.2.
periodontal therapy
Inter and intra-examiner calibrations were performed prior to
initiation of the study. Inter-examiner reliability was determined by having each examiner make dual measurements
along with those of the Project Director on at least 2 patients,
and to remediate any measurement differences >1 mm or 1
score. Intra-examiner reliability was determined by taking
repeated measurements from the same pocket at the calibration session and on about 20% of the cases (randomly
determined) during the study.
Prior to any treatment, baseline clinical indices were
recorded, and then GCF samples were collected. The clinical
recordings included plaque index (PI),18 gingival index (GI),19
probing depth (PD), and clinical attachment level (CAL).
Clinical measurements performed on six sites per
tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual,
archives of oral biology 58 (2013) 391–396
mid-lingual, disto-lingual) using with a Williams periodontal
probe (Nordent Manufacturing Inc., Elk Grove Vilage, IL, USA)
calibrated in millimetres. The deepest six pockets of each
subject were chosen for clinical recordings and GCF sampling.
Therefore, for obtaining GCF samples, 72 (12 CP patients 6
sites) pockets were sampled in CP group, 78 (13 AgP
patients 6 sites) pockets were sampled in AgP group and
120 (20 participants 6) pockets were sampled in C group at
each of the test appointments.
Periodontitis patients received non-surgical periodontal
treatment consisted of oral hygiene instruction, scaling and
root planing with ultrasonic and hand instruments, which was
performed without any adjunctive therapy and completed
within 2 weeks from the beginning of the study. After 6 weeks
from baseline measurements, clinical measurements and GCF
sampling were repeated for periodontitis groups.
2.3.
analysis among the groups, One-Sample Kolmogorow–Simirnov test was performed. According to the results of this test,
non-parametric analysis was determined. The results were
shown as median and 25–75 percentiles in the parentheses
(quarters). The x2 test was used to determine whether there
was a statistical difference among the groups with respect to
gender. Wilcoxon test was used for evaluation of the
differences before and after the treatment. Mann–Whitney
U test was used for evaluation of the differences between CP
and C and also between AgP and C before and after the
treatment. The correlations between clinical indices and GCF
t-PA and PAI-2 levels were analyzed with Spearman correlation coefficient. p-Value