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...

Osteoarthritis (OA) is the most common chronic joint disease and leads to the degradation of the extracellular matrix by an imbalance between anabolic and catabolic processes. TGF-β3 (transforming growth factor beta-3) and epidermal growth factor (EGF) influence the osteochondrogenic potential of chondrocytes. In this study, we compared the expression of mediators and receptors in the TGF-β3 and EGF pathways, as well as biglycan (BGN), in healthy and diseased chondrocytes. Furthermore, we used chondrogenic progenitor cells (CPCs) for in vitro stimulation and knockdown experiments to elucidate the effects of TGF-β3 and EGF on the chondrogenic potential. Our results demonstrate that the expression of TGF-beta receptor type-1 (TGFBRI) and epidermal growth factor receptor (EGFR) is altered in diseased chondrocytes as well as in CPCs. Moreover, TGF-β3 and EGF stimulation influenced the expression levels of BGN, SRY (sex determining region Y)-box 9 (SOX9), and Runt-related transcription factor 2 (RUNX2) in CPCs. Therefore, changes in TGFBRI and EGFR expression likely contribute to the degenerative and regenerative effects seen in late stages of OA. (J Histochem Cytochem 67:117-127, 2019)

Our new albumin depletion strategy combined with high-resolution mass spectrometry can be used to effectively monitor the molecular signals of the periodontium.

BackgroundThe present study aimed to evaluate the force delivery of removable thermoplastic appliances (RTAs), modified by different sized incisal cuts, during tipping of a maxillary central incisor in palatal and vestibular direction.MethodsForty-five RTAs from three different materials (Biolon®, Erkodur®, Ideal Clear®) of the same thickness (1 mm) were used. Analysis was performed on a separated maxillary central incisor which was part of a resin model with a complete dentition. In 15 RTAs, of different material, a cut was inserted at the incisal edge of tooth 11. In 15 other appliances, the cut was extended to teeth 12 and 21. Fifteen aligners remained uncut. The experimental tooth was tipped starting from the zero position in 0.05° steps to a maximal deflection of ± 0.42° of the incisal edge in vestibular and palatal direction, after positioning the RTA onto the model.ResultsThe horizontal (Fx) and the vertical (Fz) force components were decreased by approximately half with increasing cut size. Fz values changed during palatal tipping from a weak intrusive force, for aligners without cut, to an extrusive force with increasing cut size. Compared to both other materials used (Erkodur® and Ideal Clear®), the Biolon® aligners showed significantly higher Fx and Fz values (p < 0.0001, respectively).ConclusionsRTAs modified by different sized incisal cuts show altered biomechanical properties and an inversion of the vertical force component, during tipping of a maxillary central incisor.

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