Meaningful, reliable and valid mRNA expression analyses by real-time quantitative PCR (RT-qPCR) can only be achieved, if suitable reference genes are chosen for normalization and if appropriate RT-qPCRquality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite corresponding in-vitro gene expression studies being a focus of interest for many years, no information is available for hPDL fibroblasts on suitable reference genes, which are generally used in RT-qPCR experiments to normalize variability between samples. The aim of this study was to identify and validate suitable reference genes for normalization in untreated hPDL fibroblasts as well as experiments on orthodontic tooth movement or periodontitis (Aggregatibacter actinomycetemcomitans). We investigated the suitability of 13 candidate reference genes using four different algorithms (geNorm, NormFinder, comparative ΔC q and BestKeeper) and ranked them according to their expression stability. Overall PPIB (peptidylprolyl isomerase A), TBP (TATA-box-binding protein) and RPL22 (ribosomal protein 22) were found to be most stably expressed with two genes in conjunction sufficient for reliable normalization. This study provides an accurate tool for quantitative gene expression analysis in hPDL fibroblasts according to the MIQE guidelines and shows that reference gene reliability is treatment-specific.Orthodontics and periodontology are specialties of dentistry tending to the treatment of misaligned teeth/jaws and bacterially induced inflammation of the periodontal tissues (periodontitis), respectively, with several interactive associations existing 1 . In orthodontics mechanical forces applied to the teeth result in tensile and pressure zones within the periodontal ligament (PDL) 2 . PDL fibroblasts react to this mechanical strain with an increased synthesis of proinflammatory enzymes, cytokines and chemokines 2-4 , triggering osteoclastogenesis. Bacterial toxins from periodontal pathogens in periodontitis, such as the gram-negative Aggregatibacter actinomycetemcomitans (Agac), the key pathogen in aggressive periodontitis 5 , can in a similar way stimulate PDL fibroblasts, which are thus essential both for mediating orthodontic tooth movement and bacterial periodontitis.Real-time quantitative PCR (RT-qPCR) and DNA microarray analysis are the methods of choice to analyse transcription of cellular genes 6,7 . In contrast to microarray analysis, which allows expression profiling of a high number of genes, RT-qPCR enables a precise quantification of gene expression differences in physiological, pathological and various experimental states [8][9][10] . However, a reliable RT-qPCR setup is necessary to achieve valid results. To improve quality and reproducibility of RT-qPCR experiments, Bustin et al. published the MIQE guidelines 11 in 2009, detailing the minimum information for publication of quantitative real-time PCR experiments. A view in current literature shows that m...

hPDL fibroblasts seem to play a major mediating role in the early phase of OTM with a differentiated, time-dependent regulation and expression pattern of cytokines and other mediators.

OBJECTIVES While permanent retention is today the method of choice to stabilize orthodontic treatment outcomes, recent studies have increasingly reported posttreatment changes in tooth position during permanent retention. We conducted this study to analyze changes in the anterior mandible, whether the changes follow an underlying movement pattern, and, aiming for a preventive strategy, whether any risk factors could be identified comparing findings with the pretreatment situations. METH-ODS We included 30 patients who had worn fixed Twistflex retainers (UK 3-3) extending from canine to canine in the mandible. Casts reflecting the intraoral situations before orthodontic treatment (T0), directly after completion of active therapy (T1), and 6 months later (T2) were scanned and superimposed using Imageware Surfacer software. Posttreatment changes (T2-T1) of tooth position within the retainer block were analyzed on 3D virtual models and were compared to pretreatment (T0) and treatment-related (T1-T0) findings to identify potential risk factors. RESULTS Almost all analyzed patients revealed three-dimensional changes in tooth position within the retainer block. Comparing these movements, we repeatedly found rotated retainer blocks in labio-oral direction, while the center of rotation was located at the first incisors. This pattern was associated with intercanine expansion and excessive overjet correction during orthodontic treatment. The canines underwent the most pronounced (rotational and translational) movements. CONCLUSIONS In general permanent lingual retainers are safe but in special clinical cases retainers can induce undesired tooth movement. Risk factors seem to be intercanine expansion and excessive overjet correction during orthodontic treatment. In specific cases an additional retention device might be needed. AbstractObjectives. While permanent retention is today frequently the method of choice to stabilize orthodontic treatment outcomes, recent studies have increasingly reported posttreatment changes in tooth position during permanent retention. We conducted this study to analyze such changes in the anterior mandible, whether these follow an underlying movement pattern, and, aiming for a preventive strategy, whether any risk factors could be identified by comparing findings to the pretreatment situations. Methods.We included 30 patients who had worn a fixed Twistflex retainer extending from canine to canine in the mandible. Casts reflecting the intraoral situations before orthodontic treatment (T0), directly upon completion of active therapy (T1), and 6 months into retention (T2) were scanned and superposed using Imageware Surfacer software. Posttreatment changes (T2−T1) in tooth position inside the retainer block were analyzed on 3D virtual models and were compared to pretreatment (T0) and treatment-related (T1−T0) findings to identify potential risk factors.Results. Almost all patients revealed three-dimensional changes in tooth position within the retainer block. On comparing these movements...

The human oral microbiota consists of over 700 widespread taxa colonizing the oral cavity in several anatomically diverse oral niches. Lately, sequencing of the 16S rRNA genes has become an acknowledged, culture-independent method to characterize the oral microbiota. However, only a small amount of data are available concerning microbial differences between oral niches in periodontal health and disease. In the context of periodontitis, the cytokine expression in the gingival crevicular fluid has been studied in detail, whereas little is known about the cytokine profile in hard and soft tissue biofilms. In order to characterize oral niches in periodontal health, the oral microbiota and cytokine pattern were analyzed at seven different sites (plaque (P), gingival crevicular fluid (GCF), saliva (S), tongue (T), hard palate (HP), cheek (C) and sublingual area (U)) of 20 young adults using next-generation sequencing and multiplex immunoassays. Site-specific microbial compositions were detected, which clustered into three distinct metaniches (“P-GCF”, “S-T-HP” and “C-U”) and were associated with niche-/metaniche-specific cytokine profiles. Our findings allow the definition of distinct metaniches according to their microbial composition, partly reflected by their cytokine profile, and provide new insights into microenvironmental similarities between anatomical diverse oral niches.

Non-steroidal anti-inflammatory drugs (NSAID) are used to alleviate pain sensations during orthodontic therapy but are also assumed to interfere with associated pseudo-inflammatory reactions. In particular, the effects of partially selective COX-2 inhibition over the constitutively expressed COX-1 (11:1) on periodontal cells and tissue, as induced by the NSAID meloxicam, remain unclear. We investigate possible adverse side-effects and potentially useful beneficial effects during orthodontic therapy and examine underlying cellular and tissue reactions. We randomly assigned 63 male Fischer344 rats to three consecutive experiments of 21 animals each (cone-beam computed tomography; histology/serology; reverse-transcription quantitative real-time polymerase chain reaction) in three experimental groups (n = 7; control; orthodontic tooth movement [OTM] of the first/second upper left molars [NiTi coil spring, 0.25 N]; OTM with a daily oral meloxicam dose of 3 mg/kg). In vitro, we stimulated human periodontal ligament fibroblasts (hPDL) with orthodontic pressure (2 g/cm) with/without meloxicam (10 μM). In vivo, meloxicam significantly reduced serum C-reactive protein concentration, tooth movement velocity, orthodontically induced dentine root resorption (OIRR), osteoclast activity and the relative expression of inflammatory/osteoclast marker genes within the dental-periodontal tissue, while presenting good gastric tolerance. In vitro, we observed a corresponding significant decrease of prostaglandin E/interleukin-6/RANKL(-OPG) expression and of hPDL-mediated osteoclastogenesis. By inhibiting prostaglandin synthesis, meloxicam seems to downregulate hPDL-mediated inflammation, RANKL-induced osteoclastogenesis and, consequently, tooth movement velocity by about 50%, thus limiting its suitability for analgesia during orthodontic therapy. However, its protective effects regarding OIRR and good tolerance profile suggest future prophylactic application, which merits its further investigation.