Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Metabolomic signatures distinguish extracellular vesicles from osteoclasts and odontoclasts.

AIMS: Pathological dental root resorption and alveolar bone loss are often detected only after irreversible damage. Biomarkers in the gingival crevicular fluid or saliva could provide a means for early detection; however, such biomarkers have proven elusive. We hypothesize that a multiomic approach might yield reliable diagnostic signatures for root resorption and alveolar bone loss. Previously, we showed that extracellular vesicles (EVs) from osteoclasts and odontoclasts differ in their protein composition. In this study, we investigated the metabolome of EVs from osteoclasts, odontoclasts and clasts (non-resorbing clastic cells). MATERIALS AND METHODS: Mouse haematopoietic precursors were cultured on dentine, bone or plastic, in the presence of recombinant RANKL and CSF-1 to trigger differentiation along the clastic line. On Day 7, the cells were fixed and the differentiation state and resorptive status of the clastic cells were confirmed. EVs were isolated from the conditioned media on Day 7 and characterized by nanoparticle tracking and electron microscopy to ensure quality. Global metabolomic profiling was performed using a Thermo Q-Exactive Orbitrap mass spectrometer with a Dionex UHPLC and autosampler. RESULTS: We identified 978 metabolites in clastic EVs. Of those, 79 are potential biomarkers with Variable Interdependent Parameters scores of 2 or greater. Known metabolites cytidine, isocytosine, thymine, succinate and citrulline were found at statistically higher levels in EVs from odontoclasts compared with osteoclasts. CONCLUSION: We conclude that numerous metabolites found in odontoclast EVs differ from those in osteoclast EVs, and thus represent potential biomarkers for root resorption and periodontal tissue destruction.

RANKL and RANK in extracellular vesicles: surprising new players in bone remodeling.

Receptor activator of nuclear factor kappa B-ligand (RANKL), its receptor RANK, and osteoprotegerin which binds RANKL and acts as a soluble decoy receptor, are essential controllers of bone remodeling. They also play important roles in establishing immune tolerance and in the development of the lymphatic system and mammary glands. In bone, RANKL stimulates osteoclast formation by binding RANK on osteoclast precursors and osteoclasts. This is required for bone resorption. Recently, RANKL and RANK have been shown to be functional components of extracellular vesicles (EVs). Data linking RANKL and RANK in EVs to biological regulatory roles are reviewed, and crucial unanswered questions are examined. RANKL and RANK are transmembrane proteins and their presence in EVs allows them to act at a distance from their cell of origin. Because RANKL-bearing osteocytes and osteoblasts are often spatially distant from RANK-containing osteoclasts in vivo, this may be crucial for the stimulation of osteoclast formation and bone resorption. RANK in EVs from osteoclasts has the capacity to stimulate a RANKL reverse signaling pathway in osteoblasts that promotes bone formation. This serves to couple bone resorption with bone formation and has inspired novel bifunctional therapeutic agents. RANKL- and RANK- containing EVs in serum may serve as biomarkers for bone and immune pathologies. In summary, EVs containing RANKL and RANK have been identified as intercellular regulators in bone biology. They add complexity to the central signaling network responsible for maintaining bone. RANKL- and RANK-containing EVs are attractive as drug targets and as biomarkers.

Activation of (pro)renin by (pro)renin receptor in extracellular vesicles from osteoclasts.

The (pro)renin receptor (PRR) is a multifunctional integral membrane protein that serves as a component of the vacuolar H+-ATPase (V-ATPase) and also activates (pro)renin. We recently showed that full-length PRR, found as part of a V-ATPase sub-complex, is abundant in extracellular vesicles shed by osteoclasts. Here, we tested whether these extracellular vesicles stimulate (pro)renin. Extracellular vesicles isolated from the conditioned media of RAW 264.7 osteoclast-like cells or primary osteoclasts were characterized and counted by nanoparticle tracking. Immunoblotting confirmed that full-length PRR was present. Extracellular vesicles from osteoclasts dose-dependently stimulated (pro)renin activity, while extracellular vesicles from 4T1 cancer cells, in which we did not detect PRR, did not activate (pro)renin. To confirm that the ability of extracellular vesicles from osteoclasts to stimulate (pro)renin activity was due to the PRR, the "handle region peptide" from the PRR, a competitive inhibitor of PRR activity, was tested. It dose-dependently blocked the ability of extracellular vesicles to stimulate the enzymatic activity of (pro)renin. In summary, the PRR, an abundant component of extracellular vesicles shed by osteoclasts, stimulates (pro)renin activity. This represents a novel mechanism by which extracellular vesicles can function in intercellular regulation, with direct implications for bone biology.

Actin and Actin-Associated Proteins in Extracellular Vesicles Shed by Osteoclasts.

Extracellular vesicles (EVs) are shed by all eukaryotic cells and have emerged as important intercellular regulators. EVs released by osteoclasts were recently identified as important coupling factors in bone remodeling. They are shed as osteoclasts resorb bone and stimulate osteoblasts to form bone to replace the bone resorbed. We reported the proteomic content of osteoclast EVs with data from two-dimensional, high resolution liquid chromatography/mass spectrometry. In this article, we examine in detail the actin and actin-associated proteins found in osteoclast EVs. Like EVs from other cell types, actin and various actin-associated proteins were abundant. These include components of the polymerization machinery, myosin mechanoenzymes, proteins that stabilize or depolymerize microfilaments, and actin-associated proteins that are involved in regulating integrins. The selective incorporation of actin-associated proteins into osteoclast EVs suggests that they have roles in the formation of EVs and/or the regulatory signaling functions of the EVs. Regulating integrins so that they bind extracellular matrix tightly, in order to attach EVs to the extracellular matrix at specific locations in organs and tissues, is one potential active role for actin-associated proteins in EVs.

The proteome of extracellular vesicles released by clastic cells differs based on their substrate.

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.

Clastic cells in orthodontic treatment: Translational challenges and recent advances.

OBJECTIVES: Orthodontic treatment consists of numerous appliance activations that rely on stimulation of osteoclasts at alveolar bone sites. However, the action of osteoclast-like cells on dentin ("odontoclasts") is a pathological side effect of orthodontic treatment. The aim of this article is twofold: (a) To report preliminary results from ongoing cell culture experiments to identify unique markers of dentin resorption, and (b) To discuss our work using nanoparticle tracking analysis (NTA) and exosomes for developing biological fluid-based biopsies to monitor clastic cell activity. SETTING AND SAMPLE POPULATION: Twelve healthy volunteers in permanent dentition. MATERIAL AND METHODS: For the in vitro experiments, murine clastic cell precursors were cultured on dentin or bone slices for 7 days and phage-display biopanning was used to identify molecular surface differences between osteoclasts and odontoclasts. In the human study, gingival crevicular fluid (GCF) samples were collected using different tools and analysed for protein and exosome recovery. RESULTS: Biopanning generated antibody fragments that were uniquely reactive to odontoclasts. Numerous nanoparticles in the size range of exosomes were detected in all of the human GCF samples. CONCLUSIONS: Our results support that there are molecular differences between osteoclasts and odontoclasts. Emerging technologies may allow the use of exosomes in GCF as a clinical tool to detect markers of root resorption.

Extracellular vesicle identification in tooth movement models.

Extracellular vesicles (EVs) are 30-150 nm in diameter vesicles released by cells that serve important intercellular regulatory functions. EVs include exosomes and microvesicles. Exosomes form in multivesicular bodies and are released extracellularly as the multivesicular bodies fuse with the plasma membrane. Microvesicles bud directly from the plasma membrane. Here, we examine methods that are available or emerging to detect and study EVs during orthodontic tooth movement (OTM). EV's involvement in regulating bone remodelling associated with OTM may be demonstrated by adding isolated EVs to an animal model to change the rate of tooth movement. Exosomes in multivesicular bodies might be detected by immunogold labelling of markers in sections from the tooth and jaw and detection by electron microscopy. Gingival crevicular fluid (GCF) is enriched in EVs. Detection and characterization of EVs released by osteoclasts during resorption have been described, and this information could be used to analyse EVs in OTM models. Regulatory EVs may be enriched in the GCF from teeth that are being moved or are undergoing root resorption. Emerging approaches, including nanoparticle tracking, ExoView and micro- and nanofluidics, show promise for studying EVs in the GCF. Techniques that amplify signal, including polymerase chain reaction (PCR), provide the sensitivity necessary to utilize EVs from GCF as biomarkers. Studies of the role of EVs in OTM will provide fresh insight that may identify means for enhancing OTM procedures. EVs in GCF may include biomarkers for bone remodelling during OTM, orthodontic-associated root resorption, and other dental pathologies.

Enoxacin and bis-enoxacin stimulate 4T1 murine breast cancer cells to release extracellular vesicles that inhibit osteoclastogenesis.

Enoxacin and its bone-seeking bisphosphonate derivative, bis-enoxacin, have recently captured attention as potential therapeutic agents for the treatment of cancer and bone disease. No differences in growth or survival of 4T1 murine breast cancer cells were detected at a concentration of 50 µM of enoxacin or bis-enoxacin. Growth was perturbed at higher concentrations. Both 50 µM enoxacin and bis-enoxacin stimulated increases in the number of GW/Processing bodies, but there were minimal changes in microRNA levels. Extracellular vesicles (EVs) released from 4T1 cells treated with 50 µM enoxacin or 50 µM bis-enoxacin stimulated proliferation of RAW 264.7 cells, and both significantly inhibited osteoclastogenesis in calcitriol-stimulated mouse marrow. EVs from 4T1 cells treated with enoxacin and bis-enoxacin displayed small reductions in the amount of microRNA (miR)-146a-5p and let-7b-5p. In marked contrast, miR-214-3p, which has been shown to regulate bone remodeling, was increased 22-fold and 30-fold respectively. We conclude that enoxacin and bis-enoxacin trigger the release of EVs from 4T1 cancer cells that inhibit osteoclastogenesis.

Clinical Characteristics of Localized Aggressive Periodontitis in Primary Dentition.

OBJECTIVES: Due to the low prevalence of localized aggressive periodontitis (LAP), clinical characteristics of LAP in primary dentition are derived from a few case reports/series in the literature. The goal of this study was to determine common clinical characteristics such as bone and root resorption patterns, in a series of cases with LAP in primary dentition. We hypothesize these cases present aggressive periodontal bone destruction starting mostly around first primary molars and atypical root resorption patterns. STUDY DESIGN: We have evaluated 33 LAP cases in primary dentition for pattern of bone destruction, root resorption and early exfoliation. RESULTS: Cases evaluated were aged 5-12 (mean=8.7 years). Thirty cases presented more severe bone loss on first than second molars, with relatively fast progression to second molars, altered pattern of root resorption, mostly external (n=16) and early exfoliation of primary teeth due to periodontal bone loss, rather than physiologic root resorption (n=11). CONCLUSIONS: This study showed common clinical characteristics found in LAP in primary molars, including possible initiation on first primary molars and abnormal root resorption patterns. These characteristics are important to be early identified and treated in order to prevent possible progression into the permanent dentition.

Immunoassay analysis of proteins in gingival crevicular fluid samples from resorbing teeth.

OBJECTIVE: To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous teeth and nonresorbing controls. MATERIALS AND METHODS: A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP). RESULTS: There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P > .05). IL-1RA was the only biomarker to show a significant down-regulation (P  =  .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker. CONCLUSIONS: This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.

A prospective randomized clinical trial into the capacity of a toothpaste containing NovaMin to prevent white spot lesions and gingivitis during orthodontic treatment.

BACKGROUND: White spot lesions and gingivitis represent common, yet challenging, dilemmas for orthodontists. Fluoride has shown some benefit as a protective measure against demineralization; however, this is usually insufficient for orthodontic patients with less than ideal oral hygiene. Dentifrices containing calcium sodium phosphosilicate bioactive glass (NovaMin) have been proposed to aid in prevention of white spot lesions and gingival inflammation. Thus, the purpose of this study was to determine if the use of NovaMin reduces the formation of white spot lesions and improves gingival health in orthodontic patients. METHODS: This was a prospective, double-blind, randomized controlled trial. Forty-eight patients undergoing orthodontic treatment were randomly allocated to two groups. The control group consisted of 24 patients who received over-the-counter fluoride toothpaste (Crest®), while the study group consisted of 24 patients who were given the test dentifrice (ReNew™) containing 5 % NovaMin and fluoride. Patients were followed up for 6 months on a monthly basis. Decalcification, gingival health, plaque, and bacteria levels were evaluated every 3 months. Statistical analysis was performed using both parametric and non-parametric tests to identify differences between groups at different time points. RESULTS: There were no significant differences between the groups in regard to changes in white spot lesions, plaque, or gingival health (P > 0.05). There was a trend toward improvement in white spot lesions found in subjects using Crest® at the 3-month time point; however, this was not sustained throughout the study. CONCLUSIONS: Our results indicate that a toothpaste containing NovaMin does not differ significantly compared to traditional fluoride toothpaste for improving white spot lesions and gingivitis in orthodontic patients.

Mass spectrometry analysis of gingival crevicular fluid in the presence of external root resorption.

INTRODUCTION: In this study, we used liquid chromatography-mass spectrometry (LC-MS) to investigate the differences in the composition of gingival crevicular fluid between resorbing deciduous molars and nonresorbing permanent teeth. The main goal was to identify novel biomarkers associated with root resorption. METHODS: Eleven children (4 boys, 7 girls) in the mixed dentition were selected to participate in this split-mouth design study, in which a deciduous second molar with radiographic evidence of root resorption served as the experimental site, and the permanent first molar on the contralateral quadrant was the control site. Gingival crevicular fluid was collected using absorbing strips. A total of 22 samples (11 root resorption, 11 control) were each analyzed with 1-dimensional LC-MS. The remaining samples were then pooled across the 11 patients and analyzed by 2-dimensional LC-MS. The output files were converted to mascot generic format, which can be used to perform protein identification with conventional search engines. RESULTS: The 2-dimensional LC-MS protocol was able to identify 2789 and 2421 proteins in the control and resorption pooled samples, respectively. In this population, we detected significantly upregulated and downregulated proteins in the teeth with root resorption. Interestingly, many of these proteins are characteristically found in exosomes. CONCLUSIONS: We identified novel proteins that might prove to be useful biomarkers of root resorption, individually or as part of a panel.

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.

Digital analysis of staining properties of clear aesthetic brackets.

OBJECTIVE: To analyse the staining properties of clear orthodontic brackets using a digital analysis. DESIGN: In vitro, laboratory study MATERIAL AND METHODS: A total of 500 tooth-coloured brackets from 10 brands (five ceramic and five plastic) were investigated. The cumulative discolouring effect of staining agents (tea, coffee, curry and red wine) were analysed at two consumption levels: light and heavy, based on a 6-month period of exposure. Study group brackets were immersed in the agents consecutively at 37°C. The control group was only exposed to artificial saliva. Samples were analysed digitally to obtain L*, a* and b* (lightness, red-green and yellow-blue) colour readings. Using these values, total colour change (ΔE*) at each level was also calculated. Three-way analysis of variance (ANOVA) test was used for statistical comparisons. RESULTS: L* and b* colour parameters showed significant differences (P<0·001) between different bracket groups, consumption levels and the type of exposure. However, the a* value only differed between bracket groups (P<0·001). According to the ΔE* values, ceramic brackets had less colour change than plastic brackets at the end of phase 1 for both the study and control groups. However, as consumption time increased, the rate of colour change decreased for the plastic brackets. In general, ceramic brackets demonstrated much more resistance to staining agents than plastic brackets. CONCLUSIONS: Both plastic and ceramic brackets showed changes in colour when exposed to heavy consumption of staining agents, with plastic brackets being the most affected.

Relapse of orthodontically corrected deepbites in accordance with growth pattern.

INTRODUCTION: A common orthodontic problem is a deep overbite malocclusion. Because of its high relapse tendency, it is also one of the most challenging problems to treat. To minimize relapse, the morphologic characteristics of patients need to be considered. The aim of this study was to compare deepbite relapse in 3 groups of patients categorized by vertical growth type. METHODS: The total sample included 60 patients treated at the University of Washington in Seattle, all with initial overbites greater than 50%. Data were collected from casts and cephalometric radiographs at 3 time points: pretreatment, posttreatment, and 10 years postretention. A mixed-effects model (analysis of variance) and post-hoc t tests were used for the statistical evaluations. RESULTS: The high-angle subjects showed the least deepbite relapse (0.1 ± 1.1 mm), whereas the low-angle (1.2 ± 0.9 mm) and the normal-angle (1.4 ± 1.3 mm) subjects had statistically significant relapses P <0.001. This overbite relapse might be partially due to changes in the mandibular and interincisal angles, which were also observed in these 2 groups. CONCLUSIONS: High-angle subjects tend to relapse less in overbite than do low-angle and normal-angle subjects in the long term.

Effect of tooth bleaching on shear bond strength of a fluoride-releasing sealant.

OBJECTIVE: To evaluate the effect of an in-office plus at-home bleaching protocol on shear bond strength of orthodontic buttons when using a fluoride-releasing sealant. MATERIALS AND METHODS: Extracted human molars (160) were randomly divided into bleached (n  =  80) and unbleached groups (n  =  80). The bleached group was treated with 45% carbamide peroxide for 30 minutes, followed by five applications of 20% carbamide peroxide at 24-hour intervals. After 2 weeks, lingual buttons were bonded on the teeth in both groups using either Transbond XT primer or Pro Seal sealant. The teeth were then stored in artificial saliva and subjected to shear testing at 24 hours and 3 months using a Zwick Universal Test Machine. Comparisons of mean shear bond strength values were made with the analysis of variance test. The Fisher's exact test was used to evaluate the adhesive remnant index scores. RESULTS: The analysis of variance of the 24-hour results indicated a significant difference between the four subgroups (P < .0011). Further simple t-tests indicated that the differences were significant only between bleached and unbleached subgroups (P < .0011). The 3-month results showed the mean shear bond strengths of the unbleached group using Pro Seal sealant was significantly lower than that of the other, though still greater than clinically minimal suggested bond strengths. Interestingly, 15% of the bleached teeth exhibited enamel fracture at the 3-month testing. CONCLUSION: Both Pro Seal sealant and Transbond XT primer demonstrated reliable shear bond strength values on both bleached and unbleached teeth over time.

Impact of orthodontic retainers on periodontal health status assessed by biomarkers in gingival crevicular fluid.

OBJECTIVE: To evaluate whether biomarkers of inflammation and periodontal remodeling are differentially expressed in the gingival crevicular fluid (GCF) of patients wearing different types of orthodontic retainers. MATERIALS AND METHODS: Thirty-one adult subjects (17 men and 14 women with an age range of 20 to 35 years) were allocated to three different groups. Group 1 consisted of 10 patients wearing fixed retainers, group 2 included 11 patients using lower removable retainers, and group 3 comprised 10 patients without retainers (control). Periodontal health assessment and GCF collection were carried out at two sites per subject: the lingual side of a central lower incisor and the lingual side of a lower second premolar. Aliquots from diluted GCF were screened for the presence of biomarkers using a microarray technique. RESULTS: Group 1 patients exhibited a higher percentage of sites with visible plaque in the incisor region than the other groups (P = .03); no differences were noted in gingival bleeding and probing depths. The median concentrations (pg/mL) of interferon-gamma and interleukin-10 were significantly higher in the premolar sites of patients in group 2 (P = .01 and P = .04, respectively), whereas the concentration of matrix metalloproteinase-9 was significantly higher at the incisors of patients wearing fixed retainers (P = .02). A significant difference between the two sites was seen only in group 2. CONCLUSIONS: The presence of different orthodontic retainers may promote specific alterations in the GCF composition. With retention periods potentially becoming longer, this finding may be of clinical significance.