Nitric oxide in human gingival crevicular fluid after orthodontic force application

Nitric oxide (NO) is involved in bone remodelling and has been shown to play a role in regulating the rate of orthodontic tooth movement (OTM) in rat models. In humans, however, the role of NO in OTM remains less clear. In this study, NO concentration in gingival crevicular fluid (GCF) was measured in patients undergoing orthodontic treatment. Thirteen male participants (ages 11–18 years) planned for non-extraction fixed orthodontic therapy were recruited. Samples of GCF were collected from each maxillary central incisor and first and second molar immediately before (T0), 1 h after (T1), and 3–4 days after (T2) application of light orthodontic forces. The maxillary second molars were not included in the appliance and served as controls. Measureable NO levels were consistently obtained from all sampled sites. Total NO levels showed significantly higher NO levels (p < 0.05) at T1 at the buccal surfaces of the central incisors when compared to the first and second molars. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the periodontal ligament tissues of human teeth during OTM are affected by a force gradient and the magnitude of the applied force.     

Differences in the gingival crevicular fluid composition between adults and adolescents undergoing orthodontic treatment

Objectives:To investigate differences in the gingival crevicular fluid (GCF) composition between adolescent and adult patients undergoing orthodontic treatment with fixed appliances.Materials and Methods:Ten adolescents (14.4 ± 1.43) and 10 adults (28.5 ± 7.83) with Class I malocclusions and minor upper incisor crowding were allocated to two different age groups. Brackets were bonded only in the upper arch over the 20-week period of the experiment. Samples of GCF were collected from the labial sides of the upper incisors (experimental sites) and lower incisors (control sites) of each subject at five time points. Aliquots from diluted GCF were screened for the presence of receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), interleukin-1 (IL-1), interleukin-1 receptor antagonist (IL-1RA), and metalloproteinase-9 (MMP-9) using a microarray technique. The values were statistically analyzed.Results:In adults, the ratio of IL-1 to IL-1RA decreased significantly (P  =  .033) in experimental sites 3 weeks after appliance placement and first archwire activation. In adolescents, the ratio of RANKL to OPG peaked 6 weeks after the insertion of the first rectangular archwire. This ratio peak found in adolescents was a consequence of a decrease in the mean concentration of OPG. No significant changes over time were observed in the concentration of MMP-9.Conclusion:This study demonstrates age trends in the GCF levels of IL-1, IL-1RA, RANKL, and OPG that may be used to track differences in tissue response between adults and adolescents undergoing orthodontic treatment.     

Effects of aging on RANKL and OPG levels in gingival crevicular fluid during orthodontic tooth movement

OBJECTIVES: To compare the levels of the receptor activator of NFkB ligand (RANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) during orthodontic tooth movement in juvenile and adult patients. DESIGN: Fifteen juveniles and 15 adults served as subjects. GCF was collected from the distal cervical margins of the experimental and control teeth at 0, 1, 24, and 168 h after application of a retracting force. Enzyme-linked immunosorbent assay kits were used to determine RANKL and OPG levels in the GCF samples. RESULTS: The amount of tooth movement for juveniles was larger than for adults after 168 h. Further, after 24 h RANKL levels were increased and those of OPG decreased in GCF samples from the compression side during orthodontic tooth movement in both juveniles and adults. The RANKL/OPG ratio in GCF from adult patients was lower than that in the juvenile patient samples. CONCLUSION: Our results suggest that the age-related decrease in amount of tooth movement may be related to a decrease in RANKL/OPG ratio in GCF during the early stages of orthodontic tooth movement.     

Cytokine expression pattern in compression and tension sides of the periodontal ligament during orthodontic tooth movement in humans

Orthodontic tooth movement is achieved by the remodeling of periodontal ligament (PDL) and alveolar bone in response to mechanical loading and is believed to be mediated by several host mediators, such as cytokines. By means of real-time polymerase chain reaction (PCR), we studied the pattern of expression of mRNA encoding several pro- and anti-inflammatory cytokines in relation to several extracellular matrix and bone remodeling markers, in tension (T) and compression (C) sides of the PDL of human teeth subjected to rapid maxillary expansion. The PDL of normal teeth was used as a control. The results showed that both T and C sides exhibited significantly higher expression of all targets when compared with controls, except for type I collagen (COL-I) and tissue inhibitor of metalloproteinase-1 (TIMP-1) on the C side. Comparing C and T sides, the C side exhibited higher expression of tumor necrosis factor-alpha (TNF-alpha), receptor activator of nuclear factor-kappaB ligand (RANKL), and matrix metalloproteinase-1 (MMP-1), whereas the T side presented higher expression of interleukin-10 (IL-10), TIMP-1, COL-I, osteoprotegerin (OPG), and osteocalcin (OCN). The expression of transforming growth factor-beta (TGF-beta) was similar in both C and T sides. Our data demonstrate a differential expression of pro- and anti-inflammatory cytokines in compressed and stretched PDL during orthodontic tooth movement.     

MicroRNA-34 expression in gingival crevicular fluid correlated with orthodontic tooth movement

ObjectivesTo explore the expression of miR-34a and its effect on expression of matrix metalloproteinases (MMPs) during orthodontic tooth movement (OTM).Materials and MethodsTwenty patients, age 12–18 years old, who underwent orthodontic treatment were enrolled. The expression of miR-34a and MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-14) were detected in gingival crevicular fluid by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction at different time points. The miR-34a mimics or inhibitors were transfected into human periodontal ligament (hPDL) cells, and the MMP expression was measured by ELISA.ResultsThe miR-34 expression in GCF on both the tension and pressure sides after orthodontic treatment were significantly downregulated, while the levels of MMPs were significantly upregulated compared with baseline level. The levels of miR-34 and MMPs returned to baseline level 3 months after orthodontic treatment. The expression of miR-34 was negatively correlated with the expression of MMP-2, MMP-9, and MMP-14. After transfection with miR-34, the MMP-2, MMP-9, and MMP-14 expression by hPDL cells were significantly downregulated compared with miR-control and miR-34 inhibitor.ConclusionsDownregulated miR-34 expression was positively correlated with MMP-2, MMP-9, and MMP-14 expression. The miR-34a transfection into hPDL cells inhibited expression of MMPs. The results suggest that miR-34a is involved in expression of MMPs during OTM.     

Cytokines in crevicular fluid and orthodontic tooth movement

This review aimed to evaluate studies on cytokines in the gingival crevicular fluid (GCF) during orthodontic treatment, summarizing the regulation patterns of the most commonly studied cytokines and exploring their clinical implications. To achieve this, a number of key databases were searched using MESH terms and free text terms. An additional search was made by reference tracking. The procedures suggested by the QUOROM statement were followed. Data from the included studies were extracted into orthodontic mechanics, GCF sampling/handling methods, and cytokine measurements. From the 85 relevant studies identified, 23 studies could be included. Common drawbacks consisted mainly of inadequacies in the study design (e.g. short duration and small number of study subjects). The most consistent result was a peak of cytokine levels at 24 h. Associations existed between prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) and pain, velocity of tooth movement, and treatment mechanics. Interleukin-1β and PGE₂ showed different patterns of up-regulation, with IL-1β being more responsive to mechanical stress and PGE₂ more responsive to synergistic regulation of IL-1β and mechanical force. The results might be taken to support, at the cellular level, the use of light continuous forces for orthodontic treatment.     

The effects of orthodontic tooth movement on the glycosaminoglycan components of gingival crevicular fluid

Abstract In this study, gingival crevicular fluid (GCF) was collected from around a canine tooth, in children, before and during orthodontic tooth movement. The aim was to identify and quantify the glycosaminoglycan (GAG) components of GCF and relate them to tooth movement, gingival inflammation, plaque accumulation, pocket probing depth and GCF volume recorded at the site of sampling. GAG in GCF samples, collected for a 15-min period into microcapillary tubes, were separated electrophoretically, stained with Alcian blue and quantified using a laser densitometer. 2 GAG components of hyaluronic acid (HA) and chondroitin sulphate (CS) were identified. The increase in GCF volume during orthodontic tooth movement was only partly due to increased gingival inflammation, GAG levels varied with different types of orthodontic tooth movement. In GCF, levels of CS, in particular, may reflect the changes in the deeper periodontal tissues which could be monitored during orthodontic tooth movements.     

The subgingival microflora and gingival crevicular fluid cytokines in refractory periodontitis

Abstract Refractory periodontitis manifests as a rapid, unrelenting, progressive loss of attachment despite the type and frequency of therapy. This study examined possible relationships between cytokine levels in gingival crevicular fluid (GCF), occurrence of specific periodontopathic microflora. and disease activity in patients with refractory periodontitis. Refractory periodontitis patients (7 male and 3 female) were selected on the basis of history and longitudinal clinical observations. In each patient. 2 teeth with pocket depths greater than 6 mm were selected and individual acrylic stents were fabricated with reference grooves for each site. The sites were examined at both baseline and 3 months later. The pattern and amount of alveolar bone resorption were assayed by quantitative digital subtraction radiography. Pocket depth and attachment loss were measured with a Florida Probe. The gingival index was measured at 4 sites around each sample tooth. Sites were divided into active sites (2.1 mm loss of attachment in 3 months) or inactive sites (2.0 mm loss of attachment in 3 months). The distribution and prevalence of the predominant microflora in active and inactive sites were compared using anaerobic culture and indirect immunofluorescence. Interleukin-1β, 2, 4, 6 and tumor necrosis factor-α (TNF-α) levels in gingival crevicular fluid (GCF) were quantified by ELISA. Prevotella intermedia and Eikenella corrodens significantly decreased in inactive sites but remained the same in active sites after 3 months. The active sites revealed significantly higher GCF levels of IL-2 and IL-6 than inactive sites at both baseline and at 3 months. IL-1β was also significantly greater in active sites than in inactive sites at 3 months. Alveolar bone loss in active sites correlated with increased GCF levels of IL-1β and 1L-β. These results suggest that GCF levels of IL-1β, IL-2 and IL-6 and P. intermedia and E. corrodens in subgingival plaque may serve as possible indicators of disease activity in refractory periodontitis.     

Effect of orthodontic force on expression levels of ten cytokines in gingival crevicular fluid

Various types of inflammatory mediators are involved in the cascade of biological events behind tissue remodeling allowing orthodontic tooth movement. This split-mouth longitudinal study aimed to evaluate the gingival crevicular fluid (GCF) levels of ten cytokines, IL-6, IL-8, IL-10, IL-13, IL-17, IFN-γ, GM-CSF, MCP-1, MIP-1β and TNF-α, during initial orthodontic treatment. The sample comprised 15 healthy patients (9 males and 6 females, 13.9 ± 2.5 years). The lower (test) incisors were moved using fixed appliance carrying a 0.014-inch nickel titanium wire, whereas the upper (control) incisors were bonded without any force. The GCF was collected from the test and control teeth before fixed appliance mounting (baseline) and after 1, 7 and 21 days. In 6 sites per tooth, from canine to canine, periodontal conditions were defined as the percentage of sites with visible plaque and bleeding on probing. The total GCF cytokines levels were quantified using multianalysis Luminex technology. Throughout the experimental term, and for both test and control teeth, the mean percentage of sites with visible plaque and bleeding on probing were generally below 25% and 15%, respectively, although variability was also seen. In the test teeth, the GCF levels of all the cytokines remained constant throughout the experimental term. On the contrary, significant reductions were seen in the control teeth for each cytokine. Moreover, significantly greater levels of IL-6, GM-CSF, MCP-1 and TNFα were seen in the test teeth as compared to the control teeth at 7 days. The reasons for the differential behavior in the levels of all the investigated cytokines between the test and control teeth may be related to the presence of orthodontic forces and/or subclinical tissue inflammation. Further investigation is needed to elucidate potential roles for these biomarkers in the tissue remodeling incident to orthodontic tooth movement.     

Effect of orthodontic forces on cytokine and receptor levels in gingival crevicular fluid: a systematic review

This systematic review aimed to generate evidence on role of potent markers of inflammation [cytokines, chemokines, their associated receptors and antagonists] following the application of orthodontic forces. Subsequent to registration with PROSPERO, literature search followed a predetermined search strategy to key databases along with hand search (HS). Seventy-seven articles from PubMed (P), 637 from Scopus (S), 51 from Embase (E), and 3 from hand search (HS) were identified. A total of 39 articles were shortlisted that met strict inclusion and exclusion criteria and quality assessment. Each study was evaluated for participant characteristics, study design, oral hygiene regimen, and gingival crevicular fluid (GCF) handling. Among these studies, biomarkers in the order of frequency were interleukin (IL)-1β (N = 21), tumor necrosis factor (TNF)-α (N = 10), IL-8,IL-6(N=8), receptor activator of nuclear factor kappa-B ligand (RANKL) (N = 7), monocyte chemoattractant protein (MCP)-1 (N = 3), IL-2 (N=4), IL-4, IL-10, RANTES (N = 2), IL-1, IL-5, IL-1α, IP-10, osteopontin (OPN) (N = 1) and receptors and their antagonists in the order of osteoprotegerin (OPG) (N = 8), IL-1RA (N = 5), and RANK (N = 1). Results revealed an immediate release of inflammatory bone-resorptive mediators, IL-1β and TNF-α, where IL-1β increased as early as 1 min to 1 h reaching peak at 24 h while TNF-α increased at 1 h or 1 day. This was accompanied by a fall in bone-protective mediator (OPG) levels at 1 h and 24 h after orthodontic force application. Continuous forces were accompanied by a decrease in mediator levels after attaining peak levels (most commonly at 24 h) while repeated activations in interrupted force upregulated their secretion. Significant correlations of IL-1β levels with pain intensity, rate of orthodontic tooth movement (OTM) and of activity index (AI) (IL-1β/IL-1RA) with velocity of tooth movement and growth status of individuals have also been deduced. A greater AI and RANKL/OPG ratio was seen in juveniles as compared to adults or non-growers that were associated with faster rate of OTM in juveniles. None of the studies addressed the effect of estrous cycle in female subjects. Lack of homogeneity in several parameters calls for a better controlled research on the biology of OTM.

Electronic supplementary material

The online version of this article (doi:10.1186/s40510-014-0065-6) contains supplementary material, which is available to authorized users.     

Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro

OBJECTIVE: To determine the levels of the receptor activator of NFkB ligand (RANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) during orthodontic tooth movement. A second objective was to investigate the effect of compression force on RANKL and OPG production from human periodontal ligament (hPDL) cells. DESIGN: Ten adolescent patients were included. GCF was collected at the distal cervical margins of the experimental and control teeth 0, 1, 24, and 168 h after the retracting force was applied. Thisin vitro study was performed to examine the secretion of RANKL and OPG from hPDL cells following a compression force (0, 0.5, 1.0, 2.0, or 3.0 g/cm(2) for 48 h). Enzyme-linked immunosorbent assay (ELISA) kits were used to determine RANKL and OPG levels in the GCF and the conditioned medium. RESULTS: GCF levels of RANKL were significantly higher, and the levels of OPG significantly lower, in the experimental canines than in the control teeth at 24 h, but there were no such significant differences at 0, 1, or 168 h. In vitro study indicated that the compression force significantly increased the secretion of RANKL and decreased that of OPG in hPDL cells in a time- and force magnitude-dependent manner. The compression-stimulated secretion of RANKL increased approximately 16.7-fold and that of OPG decreased 2.9-fold, as compared with the control. CONCLUSIONS: The results obtained suggest that the changes of amount of RANKL and OPG may be involved in bone resorption as a response to compression force.     

A cross-sectional cohort study of gingival crevicular fluid biomarkers in normal-weight and obese subjects during orthodontic treatment with fixed appliances

Objectives:To investigate the effects of obesity on biomarker levels within lower incisor gingival crevicular fluid (GCF) in subjects undergoing routine fixed appliance orthodontic treatment.Materials and Methods:This was a cross-sectional clinical cohort study. GCF was collected from normal-weight and obese subjects at completion of alignment at least 1 month after placement of 0.019 × 0.025-inch stainless-steel archwires. The primary outcome was the difference in GCF biomarker levels between groups. Secondary outcomes included differences in clinical parameters of plaque and gingival indices, unstimulated whole-mouth saliva, and GCF flow rates.Results:Thirty-eight subjects (18 male, 20 female) with a mean age of 25.6 (SD, 6.3) years and mean body mass index (BMI) of 22.6 (1.6) in normal-weight and 32.4 (2.2) kg/m² in obese groups were investigated. Apart from BMI (P < .0001), there were no statistically significant differences in essential demographics between groups. Significantly increased levels of the adipokine leptin (P < .009) and the tissue-remodeling biomarker matrix metalloproteinase-9 (MMP9; P < .020) were identified in the obese cohort. For the remainder of the biomarkers, including the RANKL bone-remodeling marker and several inflammatory markers, there were no significant differences between groups. No correlation was observed between plaque index or gingival index for any GCF biomarker for either group (P = .07–1.00).Conclusions:This study investigated the GCF biochemical profile of obese and normal-weight subjects undergoing fixed-appliance orthodontic treatment. Significantly increased levels of the adipokine leptin and the tissue-remodeling biomarker MMP9 were identified in the obese group. These data provide evidence of differences in GCF biochemistry between obese and normal-weight subjects undergoing fixed appliance orthodontic treatment.     

Interleukin-1 alpha, interleukin-8 and interferon-alpha levels in gingival crevicular fluid

Cytokines play an important role in the pathology associated with chronic inflammatory diseases. We measured the total amounts [picograms (pg)] and concentrations (pg/μl) of interleukin-1 alpha (IL-lα), interleukin-8 (IL-8) and interferonalpha (IFN-α) in 20 s gingival crevicular fluid (GCF) samples obtained from 2 diseased and 2 healthy sites in 20 subjects with periodontitis, and from 2 healthy sites in 20 subjects without disease. Both the mean amount and concentration of IL-lα were significantly higher (p < 0.001) in diseased sites compared to healthy sites in subjects with disease. The results for IL-8 and IFN-α differed depending on the method of reporting. Whereas the amount of IL-8 was significantly higher (p < 0.01) in diseased sites, the mean concentration of IL-8 was lower compared to healthy sites. The mean amount of IFN-α was similar in health and disease; however, the concentration of IFN-alpha was significantly lower in diseased sites (p < 0.001) corresponding to the significant increase in crevicular fluid volume (p < 0.001). There were no significant differences in the amount or concentrations of the 3 cytokines between healthy sites from subjects with disease and healthy sites from healthy controls. The total amounts of both IFN-α and IL-8 were correlated between healthy and diseased sites in subjects. These data suggest that, while the disease status of a site is the major determinant of the levels of these cytokines locally, subjects with high levels of IL-8 and IFN-α in healthy sites also tend to have high levels of these cytokines in diseased sites. Finally, both the concentrations and total amounts of IL-8 and IFN-α were significantly correlated in diseased sites, suggesting that levels of these two cytokines rise or fall in tandem. The combination of decreased IL-8 and decreased IFN-α concentrations at diseased sites may reflect the reduced anti-bacterial host defense activity at that site.     

Cytokines in gingival crevicular fluid of adolescents and young adults

Background/aim:  The purpose of this study was to compare the levels of the cytokines interleukin-1β (IL-1β), IL-4, and IL-8 in the gingival crevicular fluid (GCF) of adolescents and young adults.
Methods:  Twenty-five adolescents aged between 14 and 16 years (Group A) and 20 periodontally healthy young adults aged between 25 and 35 years (Group B) were selected from two private dental clinics limited to pedodontics and periodontics respectively in Piraeus Greece. All subjects were systemically healthy. Clinical examination included probing pocket depth (PPD), presence or absence of plaque, and bleeding on probing (BOP). GCF was collected from four sites per subject. IL-1β, IL-4, and IL-8, measured as total amounts (pg/30 s), were evaluated in 180 samples using a commercially available sandwich enzyme-linked immunosorbent assay.
Results:  IL-1β mean levels of Groups A and B were adjusted for BOP and PPD. Differences of IL-1β mean levels between the two age groups were statistically significant ( F  = 50.245, P  < 0.001) in favour of Group A. Adolescents showed statistically significantly lower mean levels of IL-4 than young adults in the presence of BOP ( F  = 10.690, P  = 0.001). There was no statistically significant difference between adolescents and adults for the means of IL-8 adjusted for BOP and plaque presence ( F  = 2.032, P  = 0.161).
Conclusions:  Within the limits of this study the differences reported in mean levels of IL-1β and IL-4 may be attributed to the different age status.     

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.     

正畸牙齿龈沟液中白介素-17含量的研究

目的检测正畸牙加力前、加力后压力侧和张力侧龈沟液中白介素-17（IL-17）的含量。方法选择拔除4个第一双尖牙拉尖牙远移正畸患者20例,用滤纸条法收集加力前、加力1个月后上颌尖牙压力侧及张力侧龈沟液,采用双抗夹心酶联免疫吸附法（ELISA）检测龈沟液中IL-17的浓度。结果加力前、加力后压力侧和张力侧龈沟液中IL-17的浓度有显著性差异,压力侧龈沟液中IL-17的浓度明显高于加力前和张力侧（P〈0.05）。结论正畸力作用下,压力侧龈沟液中IL-17表达高于加力前和张力侧。IL-17可能参与了正畸力诱导的破骨细胞分化和牙槽骨吸收的过程。     

Evaluation of osteocalcin and pyridinium crosslinks of bone collagen as markers of bone turnover in gingival crevicular fluid during different stages of orthodontic treatment

Abstract. Osteocalcin (Oc) and the collagen cross-links pyridinoline (Pyr) and deoxypyridinoline (dPyr) arc used as markers of bone turnover in metabolic bone diseases. The aims of this study were: 1) to establish if Oc, Pyr and dPyr can be detected in GCF and 2) using the orthodontic tooth movement model of alveolar bone resorption to evaluate GCF levels of osteocalcin and these collagen cross-links as markers of bone breakdown. Plaque, colour and bleeding indices, probing measurements and GCF samples were collected at two sites in each of 20 adolescents, during 4 stages of fixed appliance therapy: (1) prior to appliance lit. (2) post appliance fit, (3) during active retraction of the maxillary canines. (4) during retention. GCF was collected onto filter paper strips and the volume determined by weighing. An ELISA kit was used for the detection of osteocalcin, whereas Pyr and dPyr were assayed using high performance liquid chromotography (HPLC). Wilcoxon signed ranks test and Bonferroni correction revealed statistically significant increases in plaque (p= 0.012), GCF volume (p= 0.024) and osteocalcin concentration (p= 0.012). between stages 1 and 2. There were no statistically significant differences between the other variables at this stage or between any of the variables at stages 2 and 3, or between stages 3 and 4, All but 3 of the GCF samples yielded detectable osteocalcin, with large site and subject variation. The median values of osteocalcin and osteocalcin concentration of all the samples were 87. ,5 pg and 66 pg/μl, with a range of 0–1,248 pg. 0–1,572 pg/μl. The detection of osteocalein in GCF during every stage, the wide variation between subjects, and the lack of a consistent pattern related to stages of orthodontic treatment, suggests that osteocalcin may merely be a constituent of GCF associated with the developing dentition, which would reduce its potential as a marker of bone turnover in this group. None of the 16 GCF samples analysed for Pyr and dPyr gave a positive result. This study confirms that fitting an orthodontic appliance results in plaque accumulation and increased gingival inflammation, and that GCF volume is the most sensitive indicator of that inflammation. Osteocalcin was detected in GCF collected from adolescents, whereas Pyr and dPyr could not be detected. Further work is required lo establish whether GCF osteocalcin levels can be used as a marker of bone turnover, and whether improvements in the sensitivity of detecting Pyr & dPyr make further study of these promising bone markers worthwhile.