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Gingival crevicular fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis
Accepted Manuscript Title: Gingival Crevicular Fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis ¨ ¨ urk, Authors: Sema Becerik, Veli Ozgen Ozt ¨ Peter Celec, Natalia Kamodyova, Gul ¨ Atilla, Gulnur ¨ Emingil PII: DOI: Reference:
S0003-9969(17)30209-1 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.06.032 AOB 3935
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Archives of Oral Biology
Received date: Revised date: Accepted date:
25-7-2016 24-6-2017 28-6-2017
¨ urk ¨ Please cite this article as: Becerik Sema, Ozt ¨ Veli Ozgen, Celec Peter, Kamodyova Natalia, Atilla Gul, ¨ Emingil Gulnur.Gingival ¨ Crevicular Fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.06.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gingival Crevicular Fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis Sema BECERİK, DDS, PhD a,* Veli Özgen ÖZTÜRK, DDS, PhD b Peter CELEC, PhD c,d Natalia KAMODYOVAc,d Gül ATİLLA, DDS, PhDa Gülnur EMİNGİL, DDS, PhDa a Ege
University, School of Dentistry, Department of Periodontology, İzmir, Turkey
bAdnan
Menderes University, School of Dentistry, Department of Periodontology, Aydın,
Turkey. c
Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia.
d
Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia *Corresponding author: Sema Becerik, DDS, PhD Ege University, School of Dentistry, Department of Periodontology, Bornova, 35100, İZMİR, TURKEY Tel: +90 232 3881105
Fax: +90 232 3880325
E-mail: [email protected] Running title: Oxidative stress, TGM2 in chronic periodontitis
Highlights
Chronic periodontitis patients have lower GCF TGM-2 than gingivitis patients.
GCF FRAP decreases in chronic periodontitis and gingivitis when compared to health.
Patients with gingivitis have lower plasma FRAP than healthy subjects.
Lower antioxidant status plays role in the pathogenesis of chronic periodontitis.
TGM2 might not have role in gingival inflammation.
TGM2 might be associated with stabilization of the extracellular matrix and wound healing.
Objective: This study was aimed to evaluate the gingival crevicular fluid (GCF) and plasma transglutaminase-2 (TGM-2), total antioxidant capacity (TAC), total oxidant status (TOS), ferric reducing antioxidant power (FRAP) and thiobarbituric acid reactive substances (TBARS) in patients with chronic periodontal disease. Materials and Methods: Twenty patients with chronic periodontitis (CP), 20 patients with gingivitis and 20 healthy subjects were enrolled in the study. Clinical periodontal parameters including probing depth, clinical attachment level, plaque index and papillary bleeding index were recorded. GCF and plasma levels of TGM-2, TAC, TOS, TBARS and FRAP were analyzed. Results: GCF TGM-2 was significantly lower in CP group than in gingivitis patients (P=0.006). GCF FRAP in CP and gingivitis groups was significantly lower than in healthy subjects (P<0.001). Plasma FRAP level was lower in gingivitis group when compared to healthy subjects (P=0.003). There was no significant difference in GCF and plasma TAC, TOS, TBARS and plasma TGM-2 levels among the study groups (P>0.05). GCF TGM-2 level was positively correlated with GCF TAC and negatively correlated with CAL. Conclusions: Decreased FRAP in GCF and plasma indicating lower antioxidant status of CP patients might suggest the role of oxidative stress in periodontitis. GCF TGM-2 data might suggest that TGM2 is associated with stabilization of the extracellular matrix and wound healing in periodontium rather than gingival inflammation.
Abbreviations: CP, chronic periodontitis; GCF, gingival crevicular fluid; TGM-2, transglutaminase-2; TAC, total antioxidant capacity; TOS, total oxidant status, FRAP, ferric reducing antioxidant power; TBARS, thiobarbituric acid reactive substances; PD, Probing depth; CAL, Clinic attachment level; PI, Plaque index; PBI, Papillary bleeding index.
Key words: transglutaminase-2, oxidative stress, gingival crevicular fluid, chronic periodontitis
Introduction Periodontitis is an inflammatory process that causes an inflammatory/immune response to the periodontopathogenic bacteria, which may lead to tissue damage in a specific, predisposed group of the population (Kantarci, Oyaizu & van Dyke, 2003; Battino et al., 2003). Periodontitis is a multifactor phenomenon (Waddington, Moseley & Embery, 2000) whose exact mechanisms of initiation and progression is not clear. Oxidative stress plays a crucial role in the periodontal tissue damage that results from host–microbial interactions, either as a direct result of excess of reactive oxygen species (ROS) or a deficiency of the antioxidant status (Chapple, Brock, Milward, Ling & Matthews, 2007; Wei, Zhang, Wang, Yang & Chen, 2010; Baltacıoğlu et al., 2014a; Baltacıoğlu et al., 2014b). Due to the short life of ROS it is not easy to detect the amount of ROS. However, ROS-related tissue destruction can be measured by analysis of oxidative stress markers. Salivary levels of those markers are related to numerous oral diseases (Tothova et al., 2015). Lipids are sensitive to oxidative damage and the final products of lipid peroxidation, are thiobarbituric acid reactive substances (TBARS) (Del Rio, Stewart & Pellegrini, 2005; Sochashi et al., 2002).
TBARS is the major and most studied product of polyunsaturated fatty acid peroxidation, indicating the increase of oxidative stress (Del Rio, Stewart & Pellegrini, 2005; Del Rio D, Serafini M & Pellegrini, 2002). Measurement of total oxidant status (TOS) can also provide a practical method for the detection of oxidative stress (Wei, Zhang, Wang, Yang & Chen, 2010). Transglutaminase-2 (TGM2) is a multifunctional calcium- dependent enzyme that catalyzes post-translational protein modifications and lead to the formation of intra- or intermolecular lysine bonds, or polyamine incorporation into proteins (Griffin, Casadio & Bergamini, 2002). TGM2 was shown to involve in several biochemical mechanisms including wound healing, signal transduction, cell proliferation and differentiation (Telci & Griffin, 2006; Ientile, Caccamo & Griffin, 2007; Curro et al., 2014). TGM2 was also found in association with hard tissue development, matrix maturation and mineralization (Lorand & Graham, 2003). It was reported that intracellular ROS played role in the stimulation of in situ tissue TGM in Swiss 3T3 fibroblasts (Lee et al., 2003). Expression of different types of TGMs, including TGM2 was shown in the human gingival tissues (Curro et al., 2014). A positive correlation was reported between TGM2 and receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio in cultured human periodontal ligament cells (Matarese et al., 2015). Almerich-Silla et al. (2015) showed that oxidative stress levels were significantly higher in the saliva of periodontal disease group than they were in the gingivitis and healthy groups and show a linear trend associated with periodontal worsening as well as bleeding on probing. Salivary concentrations of TBARS are also related to the periodontal status in adults (Celecova et al., 2013) and in children (Tóthová et al., 2013). It has been shown that TGM2 involved in the initial phase of wound healing and inflammation and cytokines and growth factors secreted during early phases of cell injury regulate TGM2 expression (Mehta, Kumar & Kim 2010). Although ROS was shown to activate TGM in-vitro (Lee et al., 2003; Yi et al., 2004) there is no study evaluating the correlation of oxidative stress with TGM2 in periodontal diseases. We hypothesized that altered oxidative stress in periodontal inflammation might affect GCF and plasma TGM-2 levels in different periodontal status. Therefore, the aim of this study was to examine the gingival crevicular fluid (GCF) and plasma TGM-2, TBARS, ferric reducing antioxidant power (FRAP), total antioxidant capacity (TAC) and TOS levels in patients with chronic periodontitis and investigate the in-vivo
correlation of TGM2 with oxidative stress.
Materials and Methods Study population A total of 60 patients were included in this study. Patients were recruited from the Department of Periodontology, School of Dentistry, Ege University in a period of 2 years between January 2011 and April 2013. The purpose and procedures were explained to all subjects before entering the study. Written informed consent in accordance with Helsinki declaration was obtained from all participants. Ethics Committee of the Ege University School of Medicine permitted the study protocol. None of the patients had a history of systemic disease that could impair immune response (such as diabetes mellitus, immunological disorders and HIV infections) and none had received antibiotics or other medicines that could affect their periodontal status or periodontal treatment within the past 3 months. The patients included were never-smokers, non-pregnant and not alcohol or antioxidant vitamin consumers. All patients underwent radiographic examination on entering into the study. Patients were included if they presented at least 16 teeth. The patients were classified into the following three groups and the selection of the patients were performed according to the clinical and radiographic criteria suggested by the International World Workshop for a Classification of Periodontal Disease and Conditions (Armitage 1999). Chronic periodontitis (CP) CP group included 20 patients including 11 males and 9 females. The patients were between 35 to 50 years old (mean of 43.1±4.2 years). They had moderate to severe alveolar bone loss with clinical attachment level (CAL) of ≥5 mm and probing depth (PD) of ≥6 mm in several sites in whole mouth. Diagnosis of CP was made when the CAL was compatiple with the amount of plaque accumulation and there was no rapid loss of periodontal tissues. Gingivitis group The gingivitis group consisted of 9 females/11 males, their age ranged from 22 to 50 (mean age 33.0±8.9 years). The patients had differing degrees of gingival inflammation and exhibited no sites with CAL > 2 mm or radiographic evidence of alveolar bone loss. Healthy group There were 10 females/10 males in the healthy group and their age changed from 30 to 50 years (mean age 38.4 ± 9.9 years). The participants were healthy volunteers from the
Department of Periodontology. They had no signs of gingival inflammation, probing depth (PD) >3 mm, CAL >2 mm alveolar bone loss. The individuals exhibited bleeding on probing in less than 10 % of the probed sites at examination. Clinical assessment The clinical periodontal parameters including PD and CAL were measured at six sites around each tooth excluding third molars with manual Williams probe (Hu Friedy, Chicago, IL, USA). Papilla bleeding index (PBI) (Saxer & Mühlemann 1975) and Turesky modification of the Quigley-Hein plaque index (PI) (Turesky, Gilmore & Glickman 1970; Quigley & Hein 1962) were also assessed. A calibrated periodontist (S.B.) achieved all the clinical measurements. The intraexaminer reliability was high and presented by an intraclass correlation coefficient of 0.90 for PD and 0.87 for CAL measurements. GCF sampling procedures Two days after the clinical measurements, patients were recalled for GCF and blood sampling. All samples were collected in the morning between 10 AM and 11 AM. Collection of the GCF samples was performed from the buccal aspects of the mesial or distal interproximal sites of 2 single rooted teeth in all groups by the same clinician (S.B.). In the CP group, GCF samples were collected from teeth with PD≥6mm and CAL ≥5 mm. In the gingivitis group samples were collected from teeth with BOP while teeth exhibiting no BOP and PD≤ 3 mm were chosen in the healthy group. The supragingival plaque was removed from the interproximal surfaces with a sterile curette before the sampling of GCF; the teeth were dried gently by an air syringe and isolated from saliva with cotton rolls. GCF sampling was achieved with filter paper (Periopaper, ProFlow, Inc., Amityville, NY, USA). Paper strips were carefully inserted into the crevice and left there for 30 seconds and care was taken to avoid mechanical injury. Strips contaminated with blood were discarded. An electronic impedance device (Periotron 8000, ProFlow Inc., Amityville, NY, USA) was used to determine the absorbed GCF volume of each strip and placed into sterile polypropylene tubes and kept at -40°C until analyzed. Blood sampling procedures Venous blood samples were obtained before any periodontal treatment initiation by a standard venipuncture from the antecubital vein of participants. Preparation of plasma was performed by centrifugation of blood at 1500 g for 10 minutes. The plasma samples were stored at -40 °C until biochemical analyses.
Biochemical analysis Two Periopaper strip samples belonging to each subject were pooled and treated with 300 µL of phosphate buffered saline (PBS, pH 7.2). Samples were incubated for 15 min at room temperature and eluted using centrifugation before removing the Periopaper strips. Human Transglutaminase 2C Polypeptide (TGM2) analysis Commercial ELISA kit, performed according to manufacturer’s guidelines (Cusabio Biotech Co., LTD., Wuhan, China), was used to analyze the levels of Human Transglutaminase-2 C Polypeptide (TGM2) within GCF and undiluted plasma samples. Briefly, 100 μL of the standard or unknown sample was added to the assay plate and incubated for 2 hours at 37°C. The liquid was then removed and Biotin-antibody was added followed by 1 hour incubation at 37°C. The wells were washed 3 times with Wash Buffer. HRP-avidin was added and the plate was incubated for 1 hour at 37°C. The wells were washed 5 times with Wash Buffer. TMB substrate was added and the plate was incubated 30 min at 37°C. Stop solution was added and optical density at 450 nm was determined. Markers of oxidative stress and antioxidant status Total antioxidant capacity (TAC) was analyzed in all samples according to the published protocol (Erel, 2004). Twenty µL of undiluted plasma or GCF sample was mixed with 0.4 mol/L acetate buffer and incubated with ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6sulphonic acid)) oxidized using hydrogen peroxide. Absorbance was measured at 660 nm and Trolox was used as a standard. Total oxidant status (TOS) was determined similarly with a spectrophotometric method (Erel, 2005). Twenty µL of undiluted plasma and GCF samples were incubated with ferrous ion - odianisidine complex in the presence of xylenol orange in acidic medium with hydrogen peroxide as a standard. Thiobarbituric acid reactive substances (TBARS) markers of lipid peroxidation, were determined spectrofluorometricaly (Behuliak et al., 2009). Twenty µL of undiluted plasma or GCF sample were derivatized with 0.67 % thiobarbituric acid in acidic medium (95°C, 45 min). Then the dyed product was isolated with n-butanol and measured (λex. = 515 nm, λem. =535 nm). Ferric reducing antioxidant power (FRAP) was analyzed by the method of Benzie and Strain (Benzie & Strain 1996). Briefly, 200 µL of prewarmed 37°C FRAP reagent (1 volume of 3 mol/L acetate buffer, pH 3.6 + 1 volume of 10 mmol/L 2,4,6-tripyridyl-S-triazine in 40
mmol/L HCl + 1 vol of 20 mmol/L FeCl3) was mixed with 20 µL of undiluted plasma or GCF sample. Absorbance was measured at 593 nm with ferrous sulphate as the standard. Proteins were quantified using BCA protein assay kit (Sigma Aldrich, Steinheim, Germany). All measurements were conducted on a Sapphire II instrument (Tecan, Grödig, Austria). Statistical analysis Considering a difference of 0.9 µmol/g prot in mean GCF levels of the biochemical markers and accepting a power of 90%, p-value of 5% in healthy and diseased groups minimum sample size was calculated based on GCF TBARS data of a previous study (Wei, Zhang, Wang, Yang & Chen, 2010). Power calculation analysis revealed that the minimum required sample size was 18 for each group. The normality of data was analyzed by D’Agostino-Pearson omnibus normality test and statistical analysis was performed using non-parametrical techniques. The patient was used as the unit of the observation. KruskalWallis test was used to compare the clinical parameters and levels of the biochemical markers detected in the study groups. If there were significant differences (p<0.05), post-hoc 2-group comparisons were assessed with Dunn's test. In order to analyze the correlations between GCF and plasma TGM-2, MDA, FRAP, TAC and TOS levels Spearman’s rank correlation analysis was used. Spearman’s rank correlation analysis was also used to correlate GCF levels of these proteins with clinical parameters of the sampling sites and plasma levels with full mouth clinical parameters and p<0.05 was considered as significant. Results There was no significant difference among the study groups regarding age and gender (P>0.05). Clinical findings The whole mouth clinical parameters are presented in Table 1. CP groups had significantly higher mean PD scores compared to the gingivitis and healthy groups (p<0.05). CAL scores were also higher in CP group when compared to gingivitis group. CP and gingivitis groups had significantly higher PBI and PI scores compared to the healthy group (p<0.05). PBI was higher in the CP group than the gingivitis group. The clinical parameters of the sampling sites are presented in Table 2. The mean PD of sampling sites in CP was significantly higher than those of the gingivitis and healthy groups (p<0.05). CP and gingivitis groups had significantly higher PBI and PI scores compared to the H group (p<0.05). CP and gingivitis groups had significantly higher GCF volume compared to
the H group (p<0.05). GCF volume was significantly higher in CP group than in gingivitis group (p<0.05). TGM-2 and Oxidative stress markers in GCF GCF TGM-2 was significantly lower in CP group than in gingivitis group (P=0.006). GCF TGM-2 in CP group was also lower than the healthy group but the difference was not significant (P>0.05) (Figure 1a). GCF TAC was reduced in CP group when compared other groups but the difference did not reached the level of statistical significance (P>0.05) (Figure 1b). GCF TOS was also similar among the study groups (P>0.05) (Figure 1c). GCF FRAP in CP and gingivitis groups were significantly lower than that of the healthy group (P<0.001, P=0.02 respectively) (Figure 1d). GCF TBARS did not differ among the study groups (P>0.05) (Figure 1e). GCF TGM-2 was positively correlated with GCF TAC and negatively correlated with CAL. GCF TOS showed positive correlation with GCF FRAP and GCF TBARS. GCF FRAP was also positively correlated with GCF TBARS and negatively correlated with PD, CAL, PI, PBI and GCF of the sampling sites (Table 3). TGM-2 and Oxidative stress markers in Plasma There was no significant difference in plasma TGM-2 between the study groups (P>0.05) (Figure 2a). Plasma TAC showed a slight increase in the CP group when compared to other groups but the difference did not reached the level of statistical significance (P>0.05) (Figure 2b). Plasma TOS was similar among the study groups (P>0.05) (Figure 2c). Plasma FRAP was lower in gingivitis group when compared to H group (P=0.003). Plasma FRAP was also reduced in CP group when compared healthy group but the difference did not reached the level of statistical significance (P>0.05) (Figure 2d). The study groups had similar plasma TBARS concentrations (P>0.05) (Figure 2e). Plasma TAC was positively correlated with full mouth PD and PBI scores while plasma FRAP was negatively correlated with full mouth CAL. (Table 4). There was no significant correlation between GCF and plasma oxidative stress markers and TGM-2 (P>0.05).
Discussion The GCF and plasma TGM-2 and oxidative stress markers levels were analyzed in CP and gingivitis groups and compared with healthy subjects in the present study. GCF TGM-2 level was significantly lower in CP group than that of gingivitis group. GCF FRAP level was higher in the healthy group when compared to CP and gingivitis groups while plasma FRAP was lower
in gingivitis group when compared to healthy group. It is important to involve patients with gingivitis in order to understand if the pronounced differences from the healthy periodontium are originating only from gingival inflammation or can be attributed to bone resorption and attachment loss. It was reported that TGM family contribute to cross-linking reactions by functions of these enzymes in cell differentiation, wound healing, apoptosis, and keratinocyte cornified envelope formation (Telci & Griffin, 2006; Ientile, Caccamo & Griffin, 2007; Curro et al., 2014). Curro et al. (2014) demonstrated that TGMs are expressed in the human gingival tissues and TGM2 maintained similar protein and mRNA expression levels in samples from chronic periodontitis and healthy gingival tissues. In line with Curro’s study (2014), the present study also showed that TGM2 levels in CP group were similar with the healthy group in both GCF and plasma samples. The similar TGM2 levels in CP group with healthy subjects might be related to the episodic character of periodontitis (Armitage 2013). Many of the sampling sites might be in episodes of remission during sampling. On the other hand, Matarese et al. (2015) reported an increased TGM2 expression in human periodontal ligament cells from periodontitis patients compared to cells from healthy subjects and a positive correlation between TGM2 and RANKL/OPG mRNA ratio, suggesting that increase in TGM2 expression may be considered an early event in tissue changes induced by periodontal disease. In contrast to our study, increased levels of TGM2 expression was suggested to be associated with high levels of pro-inflammatory markers supporting the interaction between molecular mechanisms involved in bone remodeling and resorption (Matarese et al., 2015). On the other hand, extracellular activation of TGM2 contributes to the stabilization of the extracellular matrix and promotes cell– substrate interaction and wound healing (Telci & Griffin, 2006). It has been stated that higher TGM2 could be dependent on increased levels of fibroblast proliferation and the intensified resistance of ECM to degrading enzymes (Asioli et al., 2011). Generally, high rate of TGM2 expression should be involved in the tissue response leading to the formation of specific bonds, stabilizing protein aggregates (Aeschlimann & Paulsson 1994). In this respect, healing processes have been associated with high level of TGM2 (Fisher at al., 2009). In normal and transformed cells, TGM2 is able to promote the cell–ECM interaction, and fibroblasts possessing high TGM2 levels show increased cell attachment and spreading, whereas TGM2-deficient fibroblasts exhibit decreased adhesion and spreading (Gross, Balklava & Griffin 2003). Taken together, the
higher GCF TGM-2 levels of gingivitis group than CP group while it was similar with healthy subjects might be related to the functions of TGM2 in wound healing rather than its role in gingival inflammation. It is a well-known fact that all cases of untreated gingivitis do not inevitably progress to periodontitis (Armitage 2013). It might be speculated that TGM2 might cause resistance to periodontal breakdown or favor the tissue repair in gingivitis. Moreover, previous experimental studies (Haroon et al., 2001; Buemi et al., 2004) showed that TGM2 is directly involved in the process of angiogenesis. The interaction of angiogenesis and bone formation could be explained by the presence of osteogenic cells, which were observed to arise from pericytes adjacent to small blood vessels in connective tissue (Long, Robinson, Ashcraft & Mann, 1995; Reilly, Seldes, Luchetti & Brighton 1998 ). ROS were shown to act as an important second messenger in various intracellular cell signaling in mammalian cells (Rhee, 1999). ROS is required for the activation of various signaling molecules including NF-KB, phospholipase A2 and D, MAP kinase and p70S6k in response to agonists (Rhee, 1999; Min, Kim & Exton, 1998; Bae et al., 1999). It has been reported that TGM activity is regulated by respective concentrations of intracellular calcium (Zhang, Lesort, Guttmann & Johnson, 1998) and guanosine triphosphate, in addition ROS are involved in the increase of intracellular calcium (Lee et al., 2000). Lee et al (2003) reported that intracellular ROS mediate the activation of TGM by agonists such as lysophosphatidic acid and transforming growth factor- eta. In the present study only GCF TAC levels showed positive correlation with GCF TGM-2 levels while no correlation was found between other oxidative stress markers analyzed and TGM2. Our data are limitedly supported by the previous in-vitro studies (Lee et al., 2000; Lee et al 2003). Assays of total oxidant/antioxidant status have an advantage of allowing analysis of the combined effectiveness of contributing species, which may be greater than the sum of the effects of the individual antioxidants (Chapple, Brock, Milward, Ling & Matthews, 2007). In the present study, GCF TAC was lower in the CP group than the healthy group, but the difference did not reach statistical significance. Toker et al. (Toker, Akpınar, Aydın & Poyraz, 2012) also found no differences in GCF-TAC between CP and healthy controls at baseline and 6 weeks after periodontal therapy. On the other hand Chapple et al (Chapple et al., 1996) reported that periodontitis might be associated with reduced local antioxidant defense and TAC in CP might reflect increased oxygen radical activity during periodontal inflammation locally and systemically. Also no difference in plasma TAC between CP and healthy groups
was found in the present study which is in agreement with others (Chapple, Brock, Milward, Ling & Matthews, 2007; Bostanci, Toker, Senel, Ozdemir & Aydin, 2014) while some studies showed decrease in plasma TAC in periodontitis compared to healthy controls (Baltacıoğlu et al., 2014b; Brock, Butterworth, Matthews & Chapple, 2004). FRAP assay is another way for the detection of antioxidant status and primarily measures the non-protein total antioxidant capacity (Kamodyová, Tóthová & Celec, 2015). To the best of our knowledge this is the first study evaluating GCF FRAP in periodontal disease. Besides the similar levels of GCF TAC among the study groups, GCF FRAP was found higher in CP and gingivitis groups when compared to healthy group. Within the limits of the present study FRAP assay but not TAC might indicate the reduced local antioxidant capacity in gingival inflammation. GCF and plasma TOS levels were similar among the study groups in the present study. In contrast, GCF and plasma TOS were shown to be higher in CP when compared to healthy controls in other studies (Wei, Zhang, Wang, Yang & Chen, 2010; Baltacıoğlu et al., 2014b). No significant difference was also detected in GCF and plasma TBARS among the study groups, which is in accordance with previous studies demonstrating no significant difference of plasma TBARS concentrations (Wei, Zhang, Wang, Yang & Chen, 2010). The conflicting results among studies might be related to the handling of the samples, methods used in detection of protein level and differences in study populations. A limitation of the present study is that only the amount of TGM-2 was analyzed, not its activity. However, the higher concentration of TGM-2 might suggest a higher activity of the enzyme. In conclusion, despite the higher levels of inflammation in CP group when compared to G group, GCF TGM-2 found lower in CP group and also no correlation was detected between TGM-2 and gingival inflammation. These findings might point out the contribution of TGM2 in the stabilization of the extracellular matrix and wound healing in periodontium, rather than gingival inflammation. In addition, decreased FRAP in GCF and plasma indicates lower antioxidant status of CP and gingivitis patients and supports the role of oxidative stress in gingival inflammation. Antioxidant capacity rather than lipid peroxidation in GCF seems to be relevant for the pathogenesis of periodontal disease. Future studies are needed to certain the activity of TGM2 in periodontal diseases.
Competing interests No conflicts of interest exist for any person or institution involved in this study. Funding This research was supported by Research Found of Ege University (Project no: 10-DIS014). Ethical approval The protocol and consent forms for the study, evaluated and approved by the Ethics Committee of the Ege University School of Medicine, were explained to the patients and control subjects. Acknowledgments The authors thank to Associated Prof Dr Timur Köse from Department of Biostatistics and Medical Informatics, School of Medicine, Ege University for his help in statistical analysis.
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Toker, H., Akpınar, A., Aydın, H. & Poyraz, O. (2012) Influence of smoking on interleukin1beta level, oxidant status and antioxidant status in gingival crevicular fluid from chronic periodontitis patients before and after periodontal treatment. Journal of Periodontal Research, 47(5), 572-577. Tóthová, L., Celecová, V. & Celec, P. (2013) Salivary markers of oxidative stress and their relation to periodontal and dental status in children. Disease Markers, 34(1), 9-15. Tóthová, L., Kamodyová, N., Červenka, T. & Celec, P. (2015) Salivary markers of oxidative stress in oral diseases. Frontiers in cellular and infection microbiology, 20(5), 73. Turesky, S., Gilmore, N.D. & Glickman, L. (1970) Reduced plaque formation by the chloromethyl analogue of vitamin C. Journal of Periodontology, 41, 41-43. Waddington, R. J., Moseley, R. & Embery, G.( 2000) Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases. Oral Disease 6(3), 138–151. Wei, D., Zhang, X. L., Wang, Y. Z., Yang, C. X. & Chen, G. (2010) Lipid peroxidation levels, total oxidant status and superoxide dismutase in serum, saliva and gingival crevicular fluid in chronic periodontitis patients before and after periodontal therapy. Australian Dental Journal, 55(1), 70-78. Yi, S. J., Choi, H. J., Yoo, J. O., Yuk, J. S., Jung, H.I., Lee, S. H., et al. (2004) Arachidonic acid activates tissue transglutaminase and stress fiber formation via intracellular reactive oxygen species. Biochemical and biophysical research communications, 325(3), 819-26. Zhang, J., Lesort, M., Guttmann, R. P. & Johnson, G. V. W. (1998) Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. The Journal of biological chemistry, 273(4), 2288–2298.
Figure legends: Figure 1: Levels of TGM-2 and Oxidative stress markers in GCF
1a) GCF TGM-2 levels of the study groups **: Different from the G group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.006). 1b) GCF TAC levels of the study groups 1c) GCF TOS levels of the study groups 1d) GCF FRAP levels of the study groups **: Significant difference from H group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.001). *: Different from the H group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.02). 1e) GCF TBARS levels of the study groups
Figure 2: Levels of TGM-2 and Oxidative stress markers in Plasma
2a) Plasma TGM-2 levels of the study groups 2b) Plasma TAC levels of the study groups 2c) Plasma TOS levels of the study groups. 2d) Plasma FRAP levels of the study groups *: Different from H group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.003). 2e) Plasma TBARS levels of the study groups
Figure 1: Levels of TGM-2 and Oxidative stress markers in GCF 1a
1b
1c
1d
1e
Figure 2: Levels of TGM-2 and Oxidative stress markers in Plasma 2a
2c
2d
2b
2d
Table 1: Fullmouth clinical parameters of the study groups [median (min-max)].
Clinical Parameters
CP
Gingivitis
Healthy
PD (mm)
5.4 (3.8-7.2) *†
2.8 (2-2.9) *
1.5 (1.2-1.9)
CAL (mm)
5.6 (4.1-8.5) †
0.6 (0-1.4)
-
PI
3.5 (2.5-5) *
3 (2.5-4.6) *
1.5 (1-2)
PBI
2.9 (2.5-4) *†
2.3 (1.8-2.6) *
0.6 (0-1.4)
* Significant difference from healthy group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05). † Significant difference from G group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05).
Table 2: The clinical parameters of the sampling sites [median (min-max)].
Clinical parameters
CP
Gingivitis
Healthy
PD (mm)
5.5(5-8) * †
2.5 (2-3) *
1.5 (1-2)
CAL (mm)
5.9(5-8)
-
-
PI
3 (2-5) *
3 (1-5) *
1 (0-2)
PBI
3(2-4) *†
2 (1-3) *
0.5 (0-1)
GCF (µl)
0.72 (0.35-0.9) * †
0.48 (0.35-0.9) *
0.11 (0.05-0.22)
* Significant difference from healthy group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05). † Significant difference from G group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05).
Table 3: Correlation of the oxidative stress markers and TGM-2 in GCF and clinical parameters of the sampling sites.
TGM2
TOS
P<0.001
P<0.001
R=0,575
P=0.604
P<0.001
P<0.001
R=0.785
P=0.604
P<0.001
P<0.001 R=-0.394
PD
GCF
R=0.575
P=0.014
FRAP
PBI
R=0.785
R=0.322
TBARS
PI
TBARS
P=0.014
TOS
CAL
FRAP
R=0.322
TGM2
TAC
TAC
P=0.002 R=-0.380
R=-0.373
P=0.003
P=0.003 R=-0.279 P=0.031 R=-0.348 P=0.006 R=-0.406 P=0.001
Table 4: Correlation of the oxidative stress markers and TGM-2 in plasma and full mouth clinical parameters.
TGM2
TAC
TOS
FRAP
R=0.587
TOS
P<0.001 R=0.587
TBARS
P<0.001
FRAP
PD
R=0.354 P=0,005 R=0.273
CAL
PBI
TBARS
P=0.035 R=0.349 P=0.006
S0003-9969(17)30209-1 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.06.032 AOB 3935
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
25-7-2016 24-6-2017 28-6-2017
¨ urk ¨ Please cite this article as: Becerik Sema, Ozt ¨ Veli Ozgen, Celec Peter, Kamodyova Natalia, Atilla Gul, ¨ Emingil Gulnur.Gingival ¨ Crevicular Fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.06.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Gingival Crevicular Fluid and plasma oxidative stress markers and TGM-2 levels in chronic periodontitis Sema BECERİK, DDS, PhD a,* Veli Özgen ÖZTÜRK, DDS, PhD b Peter CELEC, PhD c,d Natalia KAMODYOVAc,d Gül ATİLLA, DDS, PhDa Gülnur EMİNGİL, DDS, PhDa a Ege
University, School of Dentistry, Department of Periodontology, İzmir, Turkey
bAdnan
Menderes University, School of Dentistry, Department of Periodontology, Aydın,
Turkey. c
Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia.
d
Institute of Clinical and Translational Research, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia *Corresponding author: Sema Becerik, DDS, PhD Ege University, School of Dentistry, Department of Periodontology, Bornova, 35100, İZMİR, TURKEY Tel: +90 232 3881105
Fax: +90 232 3880325
E-mail: [email protected] Running title: Oxidative stress, TGM2 in chronic periodontitis
Highlights
Chronic periodontitis patients have lower GCF TGM-2 than gingivitis patients.
GCF FRAP decreases in chronic periodontitis and gingivitis when compared to health.
Patients with gingivitis have lower plasma FRAP than healthy subjects.
Lower antioxidant status plays role in the pathogenesis of chronic periodontitis.
TGM2 might not have role in gingival inflammation.
TGM2 might be associated with stabilization of the extracellular matrix and wound healing.
Objective: This study was aimed to evaluate the gingival crevicular fluid (GCF) and plasma transglutaminase-2 (TGM-2), total antioxidant capacity (TAC), total oxidant status (TOS), ferric reducing antioxidant power (FRAP) and thiobarbituric acid reactive substances (TBARS) in patients with chronic periodontal disease. Materials and Methods: Twenty patients with chronic periodontitis (CP), 20 patients with gingivitis and 20 healthy subjects were enrolled in the study. Clinical periodontal parameters including probing depth, clinical attachment level, plaque index and papillary bleeding index were recorded. GCF and plasma levels of TGM-2, TAC, TOS, TBARS and FRAP were analyzed. Results: GCF TGM-2 was significantly lower in CP group than in gingivitis patients (P=0.006). GCF FRAP in CP and gingivitis groups was significantly lower than in healthy subjects (P<0.001). Plasma FRAP level was lower in gingivitis group when compared to healthy subjects (P=0.003). There was no significant difference in GCF and plasma TAC, TOS, TBARS and plasma TGM-2 levels among the study groups (P>0.05). GCF TGM-2 level was positively correlated with GCF TAC and negatively correlated with CAL. Conclusions: Decreased FRAP in GCF and plasma indicating lower antioxidant status of CP patients might suggest the role of oxidative stress in periodontitis. GCF TGM-2 data might suggest that TGM2 is associated with stabilization of the extracellular matrix and wound healing in periodontium rather than gingival inflammation.
Abbreviations: CP, chronic periodontitis; GCF, gingival crevicular fluid; TGM-2, transglutaminase-2; TAC, total antioxidant capacity; TOS, total oxidant status, FRAP, ferric reducing antioxidant power; TBARS, thiobarbituric acid reactive substances; PD, Probing depth; CAL, Clinic attachment level; PI, Plaque index; PBI, Papillary bleeding index.
Key words: transglutaminase-2, oxidative stress, gingival crevicular fluid, chronic periodontitis
Introduction Periodontitis is an inflammatory process that causes an inflammatory/immune response to the periodontopathogenic bacteria, which may lead to tissue damage in a specific, predisposed group of the population (Kantarci, Oyaizu & van Dyke, 2003; Battino et al., 2003). Periodontitis is a multifactor phenomenon (Waddington, Moseley & Embery, 2000) whose exact mechanisms of initiation and progression is not clear. Oxidative stress plays a crucial role in the periodontal tissue damage that results from host–microbial interactions, either as a direct result of excess of reactive oxygen species (ROS) or a deficiency of the antioxidant status (Chapple, Brock, Milward, Ling & Matthews, 2007; Wei, Zhang, Wang, Yang & Chen, 2010; Baltacıoğlu et al., 2014a; Baltacıoğlu et al., 2014b). Due to the short life of ROS it is not easy to detect the amount of ROS. However, ROS-related tissue destruction can be measured by analysis of oxidative stress markers. Salivary levels of those markers are related to numerous oral diseases (Tothova et al., 2015). Lipids are sensitive to oxidative damage and the final products of lipid peroxidation, are thiobarbituric acid reactive substances (TBARS) (Del Rio, Stewart & Pellegrini, 2005; Sochashi et al., 2002).
TBARS is the major and most studied product of polyunsaturated fatty acid peroxidation, indicating the increase of oxidative stress (Del Rio, Stewart & Pellegrini, 2005; Del Rio D, Serafini M & Pellegrini, 2002). Measurement of total oxidant status (TOS) can also provide a practical method for the detection of oxidative stress (Wei, Zhang, Wang, Yang & Chen, 2010). Transglutaminase-2 (TGM2) is a multifunctional calcium- dependent enzyme that catalyzes post-translational protein modifications and lead to the formation of intra- or intermolecular lysine bonds, or polyamine incorporation into proteins (Griffin, Casadio & Bergamini, 2002). TGM2 was shown to involve in several biochemical mechanisms including wound healing, signal transduction, cell proliferation and differentiation (Telci & Griffin, 2006; Ientile, Caccamo & Griffin, 2007; Curro et al., 2014). TGM2 was also found in association with hard tissue development, matrix maturation and mineralization (Lorand & Graham, 2003). It was reported that intracellular ROS played role in the stimulation of in situ tissue TGM in Swiss 3T3 fibroblasts (Lee et al., 2003). Expression of different types of TGMs, including TGM2 was shown in the human gingival tissues (Curro et al., 2014). A positive correlation was reported between TGM2 and receptor activator of nuclear factor-kappa B ligand/osteoprotegerin ratio in cultured human periodontal ligament cells (Matarese et al., 2015). Almerich-Silla et al. (2015) showed that oxidative stress levels were significantly higher in the saliva of periodontal disease group than they were in the gingivitis and healthy groups and show a linear trend associated with periodontal worsening as well as bleeding on probing. Salivary concentrations of TBARS are also related to the periodontal status in adults (Celecova et al., 2013) and in children (Tóthová et al., 2013). It has been shown that TGM2 involved in the initial phase of wound healing and inflammation and cytokines and growth factors secreted during early phases of cell injury regulate TGM2 expression (Mehta, Kumar & Kim 2010). Although ROS was shown to activate TGM in-vitro (Lee et al., 2003; Yi et al., 2004) there is no study evaluating the correlation of oxidative stress with TGM2 in periodontal diseases. We hypothesized that altered oxidative stress in periodontal inflammation might affect GCF and plasma TGM-2 levels in different periodontal status. Therefore, the aim of this study was to examine the gingival crevicular fluid (GCF) and plasma TGM-2, TBARS, ferric reducing antioxidant power (FRAP), total antioxidant capacity (TAC) and TOS levels in patients with chronic periodontitis and investigate the in-vivo
correlation of TGM2 with oxidative stress.
Materials and Methods Study population A total of 60 patients were included in this study. Patients were recruited from the Department of Periodontology, School of Dentistry, Ege University in a period of 2 years between January 2011 and April 2013. The purpose and procedures were explained to all subjects before entering the study. Written informed consent in accordance with Helsinki declaration was obtained from all participants. Ethics Committee of the Ege University School of Medicine permitted the study protocol. None of the patients had a history of systemic disease that could impair immune response (such as diabetes mellitus, immunological disorders and HIV infections) and none had received antibiotics or other medicines that could affect their periodontal status or periodontal treatment within the past 3 months. The patients included were never-smokers, non-pregnant and not alcohol or antioxidant vitamin consumers. All patients underwent radiographic examination on entering into the study. Patients were included if they presented at least 16 teeth. The patients were classified into the following three groups and the selection of the patients were performed according to the clinical and radiographic criteria suggested by the International World Workshop for a Classification of Periodontal Disease and Conditions (Armitage 1999). Chronic periodontitis (CP) CP group included 20 patients including 11 males and 9 females. The patients were between 35 to 50 years old (mean of 43.1±4.2 years). They had moderate to severe alveolar bone loss with clinical attachment level (CAL) of ≥5 mm and probing depth (PD) of ≥6 mm in several sites in whole mouth. Diagnosis of CP was made when the CAL was compatiple with the amount of plaque accumulation and there was no rapid loss of periodontal tissues. Gingivitis group The gingivitis group consisted of 9 females/11 males, their age ranged from 22 to 50 (mean age 33.0±8.9 years). The patients had differing degrees of gingival inflammation and exhibited no sites with CAL > 2 mm or radiographic evidence of alveolar bone loss. Healthy group There were 10 females/10 males in the healthy group and their age changed from 30 to 50 years (mean age 38.4 ± 9.9 years). The participants were healthy volunteers from the
Department of Periodontology. They had no signs of gingival inflammation, probing depth (PD) >3 mm, CAL >2 mm alveolar bone loss. The individuals exhibited bleeding on probing in less than 10 % of the probed sites at examination. Clinical assessment The clinical periodontal parameters including PD and CAL were measured at six sites around each tooth excluding third molars with manual Williams probe (Hu Friedy, Chicago, IL, USA). Papilla bleeding index (PBI) (Saxer & Mühlemann 1975) and Turesky modification of the Quigley-Hein plaque index (PI) (Turesky, Gilmore & Glickman 1970; Quigley & Hein 1962) were also assessed. A calibrated periodontist (S.B.) achieved all the clinical measurements. The intraexaminer reliability was high and presented by an intraclass correlation coefficient of 0.90 for PD and 0.87 for CAL measurements. GCF sampling procedures Two days after the clinical measurements, patients were recalled for GCF and blood sampling. All samples were collected in the morning between 10 AM and 11 AM. Collection of the GCF samples was performed from the buccal aspects of the mesial or distal interproximal sites of 2 single rooted teeth in all groups by the same clinician (S.B.). In the CP group, GCF samples were collected from teeth with PD≥6mm and CAL ≥5 mm. In the gingivitis group samples were collected from teeth with BOP while teeth exhibiting no BOP and PD≤ 3 mm were chosen in the healthy group. The supragingival plaque was removed from the interproximal surfaces with a sterile curette before the sampling of GCF; the teeth were dried gently by an air syringe and isolated from saliva with cotton rolls. GCF sampling was achieved with filter paper (Periopaper, ProFlow, Inc., Amityville, NY, USA). Paper strips were carefully inserted into the crevice and left there for 30 seconds and care was taken to avoid mechanical injury. Strips contaminated with blood were discarded. An electronic impedance device (Periotron 8000, ProFlow Inc., Amityville, NY, USA) was used to determine the absorbed GCF volume of each strip and placed into sterile polypropylene tubes and kept at -40°C until analyzed. Blood sampling procedures Venous blood samples were obtained before any periodontal treatment initiation by a standard venipuncture from the antecubital vein of participants. Preparation of plasma was performed by centrifugation of blood at 1500 g for 10 minutes. The plasma samples were stored at -40 °C until biochemical analyses.
Biochemical analysis Two Periopaper strip samples belonging to each subject were pooled and treated with 300 µL of phosphate buffered saline (PBS, pH 7.2). Samples were incubated for 15 min at room temperature and eluted using centrifugation before removing the Periopaper strips. Human Transglutaminase 2C Polypeptide (TGM2) analysis Commercial ELISA kit, performed according to manufacturer’s guidelines (Cusabio Biotech Co., LTD., Wuhan, China), was used to analyze the levels of Human Transglutaminase-2 C Polypeptide (TGM2) within GCF and undiluted plasma samples. Briefly, 100 μL of the standard or unknown sample was added to the assay plate and incubated for 2 hours at 37°C. The liquid was then removed and Biotin-antibody was added followed by 1 hour incubation at 37°C. The wells were washed 3 times with Wash Buffer. HRP-avidin was added and the plate was incubated for 1 hour at 37°C. The wells were washed 5 times with Wash Buffer. TMB substrate was added and the plate was incubated 30 min at 37°C. Stop solution was added and optical density at 450 nm was determined. Markers of oxidative stress and antioxidant status Total antioxidant capacity (TAC) was analyzed in all samples according to the published protocol (Erel, 2004). Twenty µL of undiluted plasma or GCF sample was mixed with 0.4 mol/L acetate buffer and incubated with ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6sulphonic acid)) oxidized using hydrogen peroxide. Absorbance was measured at 660 nm and Trolox was used as a standard. Total oxidant status (TOS) was determined similarly with a spectrophotometric method (Erel, 2005). Twenty µL of undiluted plasma and GCF samples were incubated with ferrous ion - odianisidine complex in the presence of xylenol orange in acidic medium with hydrogen peroxide as a standard. Thiobarbituric acid reactive substances (TBARS) markers of lipid peroxidation, were determined spectrofluorometricaly (Behuliak et al., 2009). Twenty µL of undiluted plasma or GCF sample were derivatized with 0.67 % thiobarbituric acid in acidic medium (95°C, 45 min). Then the dyed product was isolated with n-butanol and measured (λex. = 515 nm, λem. =535 nm). Ferric reducing antioxidant power (FRAP) was analyzed by the method of Benzie and Strain (Benzie & Strain 1996). Briefly, 200 µL of prewarmed 37°C FRAP reagent (1 volume of 3 mol/L acetate buffer, pH 3.6 + 1 volume of 10 mmol/L 2,4,6-tripyridyl-S-triazine in 40
mmol/L HCl + 1 vol of 20 mmol/L FeCl3) was mixed with 20 µL of undiluted plasma or GCF sample. Absorbance was measured at 593 nm with ferrous sulphate as the standard. Proteins were quantified using BCA protein assay kit (Sigma Aldrich, Steinheim, Germany). All measurements were conducted on a Sapphire II instrument (Tecan, Grödig, Austria). Statistical analysis Considering a difference of 0.9 µmol/g prot in mean GCF levels of the biochemical markers and accepting a power of 90%, p-value of 5% in healthy and diseased groups minimum sample size was calculated based on GCF TBARS data of a previous study (Wei, Zhang, Wang, Yang & Chen, 2010). Power calculation analysis revealed that the minimum required sample size was 18 for each group. The normality of data was analyzed by D’Agostino-Pearson omnibus normality test and statistical analysis was performed using non-parametrical techniques. The patient was used as the unit of the observation. KruskalWallis test was used to compare the clinical parameters and levels of the biochemical markers detected in the study groups. If there were significant differences (p<0.05), post-hoc 2-group comparisons were assessed with Dunn's test. In order to analyze the correlations between GCF and plasma TGM-2, MDA, FRAP, TAC and TOS levels Spearman’s rank correlation analysis was used. Spearman’s rank correlation analysis was also used to correlate GCF levels of these proteins with clinical parameters of the sampling sites and plasma levels with full mouth clinical parameters and p<0.05 was considered as significant. Results There was no significant difference among the study groups regarding age and gender (P>0.05). Clinical findings The whole mouth clinical parameters are presented in Table 1. CP groups had significantly higher mean PD scores compared to the gingivitis and healthy groups (p<0.05). CAL scores were also higher in CP group when compared to gingivitis group. CP and gingivitis groups had significantly higher PBI and PI scores compared to the healthy group (p<0.05). PBI was higher in the CP group than the gingivitis group. The clinical parameters of the sampling sites are presented in Table 2. The mean PD of sampling sites in CP was significantly higher than those of the gingivitis and healthy groups (p<0.05). CP and gingivitis groups had significantly higher PBI and PI scores compared to the H group (p<0.05). CP and gingivitis groups had significantly higher GCF volume compared to
the H group (p<0.05). GCF volume was significantly higher in CP group than in gingivitis group (p<0.05). TGM-2 and Oxidative stress markers in GCF GCF TGM-2 was significantly lower in CP group than in gingivitis group (P=0.006). GCF TGM-2 in CP group was also lower than the healthy group but the difference was not significant (P>0.05) (Figure 1a). GCF TAC was reduced in CP group when compared other groups but the difference did not reached the level of statistical significance (P>0.05) (Figure 1b). GCF TOS was also similar among the study groups (P>0.05) (Figure 1c). GCF FRAP in CP and gingivitis groups were significantly lower than that of the healthy group (P<0.001, P=0.02 respectively) (Figure 1d). GCF TBARS did not differ among the study groups (P>0.05) (Figure 1e). GCF TGM-2 was positively correlated with GCF TAC and negatively correlated with CAL. GCF TOS showed positive correlation with GCF FRAP and GCF TBARS. GCF FRAP was also positively correlated with GCF TBARS and negatively correlated with PD, CAL, PI, PBI and GCF of the sampling sites (Table 3). TGM-2 and Oxidative stress markers in Plasma There was no significant difference in plasma TGM-2 between the study groups (P>0.05) (Figure 2a). Plasma TAC showed a slight increase in the CP group when compared to other groups but the difference did not reached the level of statistical significance (P>0.05) (Figure 2b). Plasma TOS was similar among the study groups (P>0.05) (Figure 2c). Plasma FRAP was lower in gingivitis group when compared to H group (P=0.003). Plasma FRAP was also reduced in CP group when compared healthy group but the difference did not reached the level of statistical significance (P>0.05) (Figure 2d). The study groups had similar plasma TBARS concentrations (P>0.05) (Figure 2e). Plasma TAC was positively correlated with full mouth PD and PBI scores while plasma FRAP was negatively correlated with full mouth CAL. (Table 4). There was no significant correlation between GCF and plasma oxidative stress markers and TGM-2 (P>0.05).
Discussion The GCF and plasma TGM-2 and oxidative stress markers levels were analyzed in CP and gingivitis groups and compared with healthy subjects in the present study. GCF TGM-2 level was significantly lower in CP group than that of gingivitis group. GCF FRAP level was higher in the healthy group when compared to CP and gingivitis groups while plasma FRAP was lower
in gingivitis group when compared to healthy group. It is important to involve patients with gingivitis in order to understand if the pronounced differences from the healthy periodontium are originating only from gingival inflammation or can be attributed to bone resorption and attachment loss. It was reported that TGM family contribute to cross-linking reactions by functions of these enzymes in cell differentiation, wound healing, apoptosis, and keratinocyte cornified envelope formation (Telci & Griffin, 2006; Ientile, Caccamo & Griffin, 2007; Curro et al., 2014). Curro et al. (2014) demonstrated that TGMs are expressed in the human gingival tissues and TGM2 maintained similar protein and mRNA expression levels in samples from chronic periodontitis and healthy gingival tissues. In line with Curro’s study (2014), the present study also showed that TGM2 levels in CP group were similar with the healthy group in both GCF and plasma samples. The similar TGM2 levels in CP group with healthy subjects might be related to the episodic character of periodontitis (Armitage 2013). Many of the sampling sites might be in episodes of remission during sampling. On the other hand, Matarese et al. (2015) reported an increased TGM2 expression in human periodontal ligament cells from periodontitis patients compared to cells from healthy subjects and a positive correlation between TGM2 and RANKL/OPG mRNA ratio, suggesting that increase in TGM2 expression may be considered an early event in tissue changes induced by periodontal disease. In contrast to our study, increased levels of TGM2 expression was suggested to be associated with high levels of pro-inflammatory markers supporting the interaction between molecular mechanisms involved in bone remodeling and resorption (Matarese et al., 2015). On the other hand, extracellular activation of TGM2 contributes to the stabilization of the extracellular matrix and promotes cell– substrate interaction and wound healing (Telci & Griffin, 2006). It has been stated that higher TGM2 could be dependent on increased levels of fibroblast proliferation and the intensified resistance of ECM to degrading enzymes (Asioli et al., 2011). Generally, high rate of TGM2 expression should be involved in the tissue response leading to the formation of specific bonds, stabilizing protein aggregates (Aeschlimann & Paulsson 1994). In this respect, healing processes have been associated with high level of TGM2 (Fisher at al., 2009). In normal and transformed cells, TGM2 is able to promote the cell–ECM interaction, and fibroblasts possessing high TGM2 levels show increased cell attachment and spreading, whereas TGM2-deficient fibroblasts exhibit decreased adhesion and spreading (Gross, Balklava & Griffin 2003). Taken together, the
higher GCF TGM-2 levels of gingivitis group than CP group while it was similar with healthy subjects might be related to the functions of TGM2 in wound healing rather than its role in gingival inflammation. It is a well-known fact that all cases of untreated gingivitis do not inevitably progress to periodontitis (Armitage 2013). It might be speculated that TGM2 might cause resistance to periodontal breakdown or favor the tissue repair in gingivitis. Moreover, previous experimental studies (Haroon et al., 2001; Buemi et al., 2004) showed that TGM2 is directly involved in the process of angiogenesis. The interaction of angiogenesis and bone formation could be explained by the presence of osteogenic cells, which were observed to arise from pericytes adjacent to small blood vessels in connective tissue (Long, Robinson, Ashcraft & Mann, 1995; Reilly, Seldes, Luchetti & Brighton 1998 ). ROS were shown to act as an important second messenger in various intracellular cell signaling in mammalian cells (Rhee, 1999). ROS is required for the activation of various signaling molecules including NF-KB, phospholipase A2 and D, MAP kinase and p70S6k in response to agonists (Rhee, 1999; Min, Kim & Exton, 1998; Bae et al., 1999). It has been reported that TGM activity is regulated by respective concentrations of intracellular calcium (Zhang, Lesort, Guttmann & Johnson, 1998) and guanosine triphosphate, in addition ROS are involved in the increase of intracellular calcium (Lee et al., 2000). Lee et al (2003) reported that intracellular ROS mediate the activation of TGM by agonists such as lysophosphatidic acid and transforming growth factor- eta. In the present study only GCF TAC levels showed positive correlation with GCF TGM-2 levels while no correlation was found between other oxidative stress markers analyzed and TGM2. Our data are limitedly supported by the previous in-vitro studies (Lee et al., 2000; Lee et al 2003). Assays of total oxidant/antioxidant status have an advantage of allowing analysis of the combined effectiveness of contributing species, which may be greater than the sum of the effects of the individual antioxidants (Chapple, Brock, Milward, Ling & Matthews, 2007). In the present study, GCF TAC was lower in the CP group than the healthy group, but the difference did not reach statistical significance. Toker et al. (Toker, Akpınar, Aydın & Poyraz, 2012) also found no differences in GCF-TAC between CP and healthy controls at baseline and 6 weeks after periodontal therapy. On the other hand Chapple et al (Chapple et al., 1996) reported that periodontitis might be associated with reduced local antioxidant defense and TAC in CP might reflect increased oxygen radical activity during periodontal inflammation locally and systemically. Also no difference in plasma TAC between CP and healthy groups
was found in the present study which is in agreement with others (Chapple, Brock, Milward, Ling & Matthews, 2007; Bostanci, Toker, Senel, Ozdemir & Aydin, 2014) while some studies showed decrease in plasma TAC in periodontitis compared to healthy controls (Baltacıoğlu et al., 2014b; Brock, Butterworth, Matthews & Chapple, 2004). FRAP assay is another way for the detection of antioxidant status and primarily measures the non-protein total antioxidant capacity (Kamodyová, Tóthová & Celec, 2015). To the best of our knowledge this is the first study evaluating GCF FRAP in periodontal disease. Besides the similar levels of GCF TAC among the study groups, GCF FRAP was found higher in CP and gingivitis groups when compared to healthy group. Within the limits of the present study FRAP assay but not TAC might indicate the reduced local antioxidant capacity in gingival inflammation. GCF and plasma TOS levels were similar among the study groups in the present study. In contrast, GCF and plasma TOS were shown to be higher in CP when compared to healthy controls in other studies (Wei, Zhang, Wang, Yang & Chen, 2010; Baltacıoğlu et al., 2014b). No significant difference was also detected in GCF and plasma TBARS among the study groups, which is in accordance with previous studies demonstrating no significant difference of plasma TBARS concentrations (Wei, Zhang, Wang, Yang & Chen, 2010). The conflicting results among studies might be related to the handling of the samples, methods used in detection of protein level and differences in study populations. A limitation of the present study is that only the amount of TGM-2 was analyzed, not its activity. However, the higher concentration of TGM-2 might suggest a higher activity of the enzyme. In conclusion, despite the higher levels of inflammation in CP group when compared to G group, GCF TGM-2 found lower in CP group and also no correlation was detected between TGM-2 and gingival inflammation. These findings might point out the contribution of TGM2 in the stabilization of the extracellular matrix and wound healing in periodontium, rather than gingival inflammation. In addition, decreased FRAP in GCF and plasma indicates lower antioxidant status of CP and gingivitis patients and supports the role of oxidative stress in gingival inflammation. Antioxidant capacity rather than lipid peroxidation in GCF seems to be relevant for the pathogenesis of periodontal disease. Future studies are needed to certain the activity of TGM2 in periodontal diseases.
Competing interests No conflicts of interest exist for any person or institution involved in this study. Funding This research was supported by Research Found of Ege University (Project no: 10-DIS014). Ethical approval The protocol and consent forms for the study, evaluated and approved by the Ethics Committee of the Ege University School of Medicine, were explained to the patients and control subjects. Acknowledgments The authors thank to Associated Prof Dr Timur Köse from Department of Biostatistics and Medical Informatics, School of Medicine, Ege University for his help in statistical analysis.
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Figure legends: Figure 1: Levels of TGM-2 and Oxidative stress markers in GCF
1a) GCF TGM-2 levels of the study groups **: Different from the G group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.006). 1b) GCF TAC levels of the study groups 1c) GCF TOS levels of the study groups 1d) GCF FRAP levels of the study groups **: Significant difference from H group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.001). *: Different from the H group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.02). 1e) GCF TBARS levels of the study groups
Figure 2: Levels of TGM-2 and Oxidative stress markers in Plasma
2a) Plasma TGM-2 levels of the study groups 2b) Plasma TAC levels of the study groups 2c) Plasma TOS levels of the study groups. 2d) Plasma FRAP levels of the study groups *: Different from H group (Kruskal Wallis test, p<0.05, Dunn’s test, P=0.003). 2e) Plasma TBARS levels of the study groups
Figure 1: Levels of TGM-2 and Oxidative stress markers in GCF 1a
1b
1c
1d
1e
Figure 2: Levels of TGM-2 and Oxidative stress markers in Plasma 2a
2c
2d
2b
2d
Table 1: Fullmouth clinical parameters of the study groups [median (min-max)].
Clinical Parameters
CP
Gingivitis
Healthy
PD (mm)
5.4 (3.8-7.2) *†
2.8 (2-2.9) *
1.5 (1.2-1.9)
CAL (mm)
5.6 (4.1-8.5) †
0.6 (0-1.4)
-
PI
3.5 (2.5-5) *
3 (2.5-4.6) *
1.5 (1-2)
PBI
2.9 (2.5-4) *†
2.3 (1.8-2.6) *
0.6 (0-1.4)
* Significant difference from healthy group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05). † Significant difference from G group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05).
Table 2: The clinical parameters of the sampling sites [median (min-max)].
Clinical parameters
CP
Gingivitis
Healthy
PD (mm)
5.5(5-8) * †
2.5 (2-3) *
1.5 (1-2)
CAL (mm)
5.9(5-8)
-
-
PI
3 (2-5) *
3 (1-5) *
1 (0-2)
PBI
3(2-4) *†
2 (1-3) *
0.5 (0-1)
GCF (µl)
0.72 (0.35-0.9) * †
0.48 (0.35-0.9) *
0.11 (0.05-0.22)
* Significant difference from healthy group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05). † Significant difference from G group (Kruskal Wallis test, p<0.05, Dunn’s test, P<0.05).
Table 3: Correlation of the oxidative stress markers and TGM-2 in GCF and clinical parameters of the sampling sites.
TGM2
TOS
P<0.001
P<0.001
R=0,575
P=0.604
P<0.001
P<0.001
R=0.785
P=0.604
P<0.001
P<0.001 R=-0.394
PD
GCF
R=0.575
P=0.014
FRAP
PBI
R=0.785
R=0.322
TBARS
PI
TBARS
P=0.014
TOS
CAL
FRAP
R=0.322
TGM2
TAC
TAC
P=0.002 R=-0.380
R=-0.373
P=0.003
P=0.003 R=-0.279 P=0.031 R=-0.348 P=0.006 R=-0.406 P=0.001
Table 4: Correlation of the oxidative stress markers and TGM-2 in plasma and full mouth clinical parameters.
TGM2
TAC
TOS
FRAP
R=0.587
TOS
P<0.001 R=0.587
TBARS
P<0.001
FRAP
PD
R=0.354 P=0,005 R=0.273
CAL
PBI
TBARS
P=0.035 R=0.349 P=0.006