Ali Gürkan

Subantimicrobial-Dose Doxycycline and Cytokine-Chemokine Levels in Gingival Crevicular Fluid

BACKGROUND The present randomized, double-masked, placebo-controlled, parallel-arm study examines the impact of adjunctive subantimicrobial-dose doxycycline (SDD) on the local inflammatory response through cytokine and chemokine levels in gingival crevicular fluid (GCF) samples from patients with chronic periodontitis. METHODS Forty-six patients with chronic periodontitis received scaling and root planing with or without adjunctive SDD. GCF samples were collected and clinical parameters including probing depth, clinical attachment level, gingival index, and plaque index were recorded every 3 months for 12 months. GCF tumor necrosis factor-α, interleukin (IL)-6, IL-4, IL-10, IL-13, IL-17, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, monocyte chemoattractant protein 1, and regulated on activated normal T-cell expressed and secreted protein levels were determined by xMAP multiplex immunoassay. RESULTS Significant improvements were observed in all clinical parameters in both groups over 12 months (P <0.0125), whereas the SDD group showed significantly better reduction in gingival index, probing depth, and gain in clinical attachment compared to the placebo group (P <0.05). Decrease in IL-6 in the SDD group was significantly higher compared to the placebo group at 6 and 9 months in deep pockets (P <0.05), whereas tumor necrosis factor-α was significantly reduced in moderately deep pockets (P <0.05). SDD resulted in a stable IL-4 and IL-10 response while reducing the monocyte chemoattractant protein 1 levels at 3 months (P <0.05). CONCLUSIONS These results show that SDD, as an adjunct to non-surgical periodontal therapy, stabilizes the inflammatory response by promoting the suppression of proinflammatory cytokines and increasing the anti-inflammatory cytokines. The chemokine activity would account for the regulation of the inflammatory response to SDD therapy.

Therapeutic efficacy of vasoactive intestinal peptide in escherichia coli lipopolysaccharide-induced experimental periodontitis in rats.

BACKGROUND The aim of the present study was to evaluate the therapeutic efficacy of vasoactive intestinal peptide (VIP), an immunoregulatory molecule, in experimental periodontitis. METHODS Sixty-three male Sprague-Dawley rats were divided into five groups: control; lipopolysaccharide (LPS); LPS + 0.1 nmol VIP; LPS + 1 nmol VIP; and LPS + 10 nmol VIP. Saline was injected into the gingiva of control rats on days 1, 3, and 5, whereas the other groups received injections of Escherichia coli LPS. VIP groups received intraperitoneal injections of relevant dosages on days 2, 4, 6, 8, and 10. The control and LPS groups were given saline instead of VIP in the same manner. On day 11, serum samples were obtained, and rats were sacrificed. Alveolar bone loss; serum levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, -10, and -18, soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), and osteoprotegerin (OPG); and the immune expression of RANKL and OPG were evaluated. RESULTS The application of VIP caused a dose-dependent decline in alveolar bone loss compared to the LPS group, but the differences were not significant (P >0.05). A reduction in the histologic findings of inflammation was observed in all VIP groups. The 1- and 10-nmol VIP groups showed significantly lower serum sRANKL and OPG levels compared to the LPS group (P <0.05). The number of positively stained vessels for endothelial OPG was greater in the 1-nmol VIP group than in the LPS group (P <0.05). CONCLUSION When periodontitis was induced by E. coli LPS, VIP downregulated the inflammatory response and inhibited alveolar bone loss, possibly by differentially regulating the tissue levels of RANKL and OPG.

Angiotensin-converting enzyme (ACE), angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1R) gene polymorphisms in generalized aggressive periodontitis

AIM Host response to periodontopathic microorganisms can be modulated by genetic factors. Accumulated evidence highlighted the role of renin-angiotensin system (RAS) in inflammatory response thus potential implication of this molecular system in the pathogenesis of periodontitis can be suggested. The present study investigated common genetic variants of molecules within the RAS family namely angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type 1 receptor (AT1R) in relation to generalized aggressive periodontitis (G-AgP). METHODS DNA was obtained from peripheral blood of 103 G-AgP patients and 100 periodontally healthy subjects. ACE I/D, AGT M235T and AT1R A1166C polymorphisms were genotyped by polymerase chain reaction and restriction fragment length polymorphism method. Chi-square, ANOVA and logistic regression were used in statistical analyses. RESULTS Both ACE I/D and AT1R polymorphisms were similar in G-AgP and healthy groups (p>0.05). G-AgP subjects exhibited decreased AGT TT genotype and T allele frequency as compared to healthy subjects (p<0.05). The same trend was also observed in the nonsmoker subgroup regarding investigated RAS polymorphisms. CONCLUSIONS Present findings suggest that AGT M235T TT genotype and T allele might be associated with decreased risk for G-AgP in Turkish population.

Matrix Metalloproteinase (MMP)-8 and Tissue Inhibitor of MMP-1 (TIMP-1) Gene Polymorphisms in Generalized Aggressive Periodontitis: Gingival Crevicular Fluid MMP-8 and TIMP-1 Levels and Outcome of Periodontal Therapy

BACKGROUND The aim of the present study is to investigate matrix metalloproteinase (MMP)-8 and tissue inhibitor of MMP-1 (TIMP-1) gene polymorphisms in generalized aggressive periodontitis (GAgP) and to assess the effects of MMP-8 and TIMP-1 genotypes on the outcomes of non-surgical periodontal therapy. METHODS Genomic DNA was obtained from peripheral blood of 100 patients with GAgP and 167 periodontally healthy controls. MMP-8 +17 C/G, -799 C/T, -381 A/G and TIMP-1 372 T/C, *429 T/G polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. Patients with GAgP received non-surgical periodontal therapy and were followed for 6 months. Clinical periodontal parameters and gingival crevicular fluid (GCF) samples were collected at baseline and at follow-up visits. GCF biomarkers were analyzed by immunofluorescence assay and enzyme-linked immunosorbent assay. RESULTS Distribution of the MMP-8 -799 C/T genotypes was significantly different between the GAgP and control groups (P <0.005). TIMP-1 372 T/C and *429 T/G genotypes in males were also significantly different between study groups (P <0.004). GCF MMP-8 levels decreased until 3 months after non-surgical therapy compared with baseline in T and G alleles, as well as G and C allele carriers (P <0.0125), whereas no significant decreased was observed in non-carriers (P >0.0125). CONCLUSION On the basis of the present findings, it can be suggested that MMP-8 -799 C/T and TIMP-1 372 T/C, *429 T/G gene polymorphisms in males may be associated with the susceptibility to GAgP in the Turkish population.

Post‐treatment effects of subantimicrobial dose doxycycline on clinical parameters and gingival crevicular fluid transforming growth factor‐β1 in severe, generalized chronic periodontitis

OBJECTIVE Present study aimed to evaluate the effect of 3-month adjunctive subantimicrobial dose doxycycline (SDD) on clinical parameters and gingival crevicular fluid (GCF) transforming growth factor-beta1 (TGF-beta(1)) levels in chronic periodontitis patients over 12 months. METHODS Thirty-five patients with severe, generalized periodontitis participated in the present randomized, placebo-controlled study. Patients received scaling and root planing (SRP) plus 3 months adjunctive SDD or placebo. Clinical measurements and GCF sampling were performed at baseline, 3, 6, 9 and 12 months. Eleven periodontally healthy subjects served as controls for GCF TGF-beta(1) analysis. RESULTS Clinical parameters of both SDD and placebo groups significantly improved during the study (P < 0.0125). SDD group exhibited significantly higher PD reduction at deep sites (baseline PD > or =7 mm) compared with placebo group at 6 months (P < 0.05). In SDD group significantly higher percentage of deep pockets resolved (PD reduction > or =3 mm from baseline) when compared with placebo group at 6 and 9 months (73.4% versus 49.7%; 79.9% versus 50.6%, respectively, P < 0.05). PD reduction > or =4 mm for deep pockets from baseline was also greater in SDD group than placebo at 6 months (53.4% versus 36.3%, P < 0.05). GCF TGF-beta(1) levels of SDD group was significantly higher than baseline (P < 0.0125) and placebo group (P < 0.017) at 3 months. CONCLUSIONS These results ensure further data for beneficial effects of adjunctive SDD therapy in the management of severe chronic periodontitis.

Renin-angiotensin gene polymorphisms in relation to severe chronic periodontitis.

AIM Evidence suggests that the ultimate product of the renin-angiotensin system (RAS), angiotensin II, exerts inflammatory actions. The present study aimed to evaluate the inter-relation between gene polymorphisms of the RAS components; angiotensin converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type-I receptor (AT1R), and severe chronic periodontitis (CP). MATERIAL AND METHODS DNA was obtained from peripheral blood of 90 CP patients and 126 periodontally healthy subjects, and the clinical parameters were recorded. ACE I/D, AGT M235T and AT1R A1166C polymorphisms were genotyped by the PCR-RFLP method. Chi-square, anova and logistic regression methods were used in statistical analyses. RESULTS The frequency of the ACE D allele was significantly lower in the CP group than the healthy group (p(corr)=0.015). CP subjects exhibited increased C allele carriage and C allele frequency of the AT1R gene (p(corr)=0.03 and p(corr)=0.03, respectively). All clinical parameters of CP patients were found to be similar in variant allele-carrying and non-carrying subjects (p>0.05). CONCLUSIONS The present findings suggest that ACE I/D and AT1R polymorphisms might be associated with susceptibility to CP but not with disease severity. The D allele of ACE I/D might be associated with decreased, whereas the C variant of AT1R A1166C might be associated with an elevated risk for CP in Turkish population.

Effect of centrifugation time on growth factor and MMP release of an experimental platelet-rich fibrin-type product

Abstract Platelet-rich fibrin (PRF) has a controlled release of growth factors due to the fibrin matrix structure. Different centrifugation protocols were suggested for PRF preparation. Since the derivation method of PRF can alter its contents, in the present study it is aimed to investigate the cell contents and transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF-AB), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and-8 release from experimental PRF-type membranes obtained with different centrifugation times at 400 gravity. Three blood samples were collected from 20 healthy non-smoker volunteers. One tube was used for whole blood analyses. The other two tubes were centrifuged at 400 g for 10 minutes (group A) or 12 minutes (group B). Each experimental PRF-type membrane was placed in Dulbecco’s Modified Eagle’s Medium (DMEM)and at 1, 24 and 72 hours, TGF-β1, PDGF-AB, VEGF, MMP-1 and -8 release amounts were analysed by enzyme-linked immunosorbent assay (ELISA). The blood cell count of membranes was determined by subtracting plasma supernatant and red blood cell (RBC) mixture from the whole blood cell counts. At 72 hours, the VEGF level of group B was statistically higher than that of group A (p = 0.040). The centrifugation time was not found to influence the release of other growth factors, enzymes and cell counts. Within the limits of the present study, it might be suggested that centrifugation time at a constant gravity has a significant effect on the VEGF levels released from experimental PRF-type membrane. It can be concluded that due to the importance of VEGF in the tissue healing process, membranes obtained at 12-minute centrifugation time may show a superior potential in wound healing.

The effect of piezoelectric surgery implant osteotomy on radiological and molecular parameters of peri‐implant crestal bone loss: a randomized, controlled, split‐mouth trial

AIM To evaluate the effect of piezoelectric surgery (PS) implant osteotomy on biochemical and radiological parameters of crestal bone (CB) loss. MATERIAL AND METHODS In this randomized, controlled, clinical study, 38 osteotomies were prepared with PS and drilling in the posterior maxilla in a split-mouth design. Implants were placed and left for non-submerged healing. Osteotomy time, insertion torque, pain perception, probing depth, and modified gingival and plaque indices were recorded. Peri-implant sulcular fluid (PISF) was collected from four sites of each implant at 2, 4, 8, 12, and 24 weeks. PISF samples were analyzed by ELISA for receptor activator of nuclear factor kappa-B-ligand (RANKL) and osteoprotegerin. CB loss was assessed on periapical radiographs at the 12th and on cone beam computed tomography (CBCT) at the 24th weeks. The influence of time and osteotomy method on biochemical and radiological parameters of CB loss employed statistical method of Brunner-Langer. RESULTS Osteotomy time for PS group was significantly longer than the drill group (P < 0.05). Pain perception that was lower in the PS than in the drill group depended on osteotomy method (P < 0.05). PS group had lower RANKL total amount than the drill group (P < 0.05). Mean CB loss on periapical radiographs at the 12th week for PS and drill groups were 0.11 and 0.18 mm, respectively (P > 0.05). At the 24th week, PS and drill groups showed 0.11 and 0.12 mm CB losses on CBCT, respectively (P > 0.05). However, CB loss values did not depend on osteotomy modality (P > 0.05). CONCLUSION PS may modify and reduce bone-destructive inflammatory response during implant osseointegration. Therefore, on the molecular level, it might be a less traumatic osteotomy modality than drilling although this was not reflected by CB loss values in the present study.

Immunohistochemical Analysis of Inducible and Endothelial Forms of Nitric Oxide Synthase in Cyclosporin A-Induced Gingival Overgrowth

BACKGROUND The contribution of nitric oxide (NO) to immune response and matrix degradation in the periodontal environment suggests a role for NO and NO-synthase (NOS) activity in the pathogenesis of cyclosporin A (CsA)-induced gingival overgrowth (GO). However, current knowledge on this topic is limited to experimental animal studies. The present study was undertaken on the basis of a hypothesis whether altered nitrite/nitrate levels in gingival crevicular fluid (GCF) and endothelial NOS (eNOS) and inducible NOS (iNOS) immunoreactivity in gingiva of CsA-treated patients contribute to the pathogenesis of CsA-induced GO. METHODS Twenty-four CsA-medicated renal transplant patients with GO (GO+; n = 12) or without GO (GO-; n = 12), 10 gingivitis, and 10 healthy subjects were included in the study. GCF samples from two proximal sites facing interdental papilla were collected, and papilla was excised. iNOS and eNOS were determined by immunohistochemistry. GCF nitrite/nitrate levels were analyzed based on the Griess reaction. RESULTS Weak iNOS immunostaining was observed in the healthy and GO- groups. In the gingivitis and GO+ groups, iNOS immunostaining significantly increased in connective tissue. Epithelial immunostaining of iNOS was localized to basal keratinocytes and the lower layer of stratum (str.) spinosum in the gingivitis group. In the GO+ group, iNOS immunostaining was differentially localized to keratinocytes of str. superficiale but considerably decreased in the str. basale. Weak eNOS immunostaining was found in the healthy and GO- groups, whereas higher immunostaining was observed in the gingivitis and GO+ groups. No intergroup differences were observed regarding nitrite/nitrate levels in GCF. CONCLUSION CsA differentially upregulated iNOS, but not eNOS, in overgrown gingiva, which may play a pivotal role in the pathogenesis of CsA-induced GO.

Gingival crevicular fluid transforming growth factor-β1 in cyclosporine and tacrolimus treated renal transplant patients without gingival overgrowth

BACKGROUND Gingival crevicular fluid (GCF) levels of transforming growth factor-beta(1) (TGF-beta(1)) have been previously investigated in relation to the pathogenesis of cyclosporine-A (CsA)-induced gingival overgrowth (GO) but no clinical data are available regarding the GCF levels of TGF-beta(1) in patients treated with tacrolimus (Tac). However, as gingival inflammation is pronounced at sites of GO and this consequently may lead to an elevation in TGF-beta(1) levels the present study aimed to evaluate gingival crevicular fluid (GCF) TGF-beta(1) levels in renal transplant patients using CsA or Tac without GO. METHODS GCF TGF-beta(1) levels were investigated in 30 renal transplant patients without GO medicated with either CsA (n=15) or Tac (n=15). Sixteen gingivitis patients and 15 periodontally healthy subjects were selected as controls. Periodontal status was evaluated by measuring probing depth, plaque index and papilla bleeding index. The TGF-beta(1) levels were analysed by enzyme-linked immunosorbent assay. RESULTS Both CsA and Tac groups had significantly elevated GCF TGF-beta(1) total amount compared to gingivitis and healthy groups (p<0.008). GCF TGF-beta(1) total amount of CsA and Tac groups was similar (p>0.008). Gingivitis and healthy groups had also similar GCF TGF-beta(1) total amount (p>0.008). CONCLUSIONS Within the limits of the present data it is unlikely that TGF-beta(1) is an exclusive mediator of CsA- or Tac-induced GO. However, pathogenesis of GO is multifactorial and contribution of TGF-beta(1) to the interrelations between cytokines and growth factors with fibrogenic potential cannot be disregarded.