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-qPCR quality standards are met. Human periodontal ligament (hPDL) fibroblasts play a major mediating role in orthodontic tooth movement and periodontitis. Despite correspondingin-vitrogene 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_qand 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.

During orthodontic tooth movement (OTM) mechanical forces trigger pseudo-inflammatory, osteoclastogenic and remodelling processes in the periodontal ligament (PDL) that are mediated by PDL fibroblasts via the expression of various signalling molecules. Thus far, it is unknown whether these processes are mainly induced by mechanical cellular deformation (mechanotransduction) or by concomitant hypoxic conditions via the compression of periodontal blood vessels. Human primary PDL fibroblasts were randomly seeded in conventional six-well cell culture plates with O₂-impermeable polystyrene membranes and in special plates with gas-permeable membranes (Lumox®, Sarstedt), enabling the experimental separation of mechanotransducive and hypoxic effects that occur concomitantly during OTM. To simulate physiological orthodontic compressive forces, PDL fibroblasts were stimulated mechanically at 2 g·cm⁻²for 48 h after 24 h of pre-incubation. We quantified the cell viability by MTT assay, gene expression by quantitative real-time polymerase chain reaction (RT-qPCR) and protein expression by western blot/enzyme-linked immunosorbent assays (ELISA). In addition, PDL-fibroblast-mediated osteoclastogenesis (TRAP⁺cells) was measured in a 72-h coculture with RAW264.7 cells. The expression of HIF-1α, COX-2, PGE2, VEGF, COL1A2, collagen and ALPL, and the RANKL/OPG ratios at the mRNA/protein levels during PDL-fibroblast-mediated osteoclastogenesis were significantly elevated by mechanical loading irrespective of the oxygen supply, whereas hypoxic conditions had no significant additional effects. The cellular–molecular mediation of OTM by PDL fibroblasts via the expression of various signalling molecules is expected to be predominantly controlled by the application of force (mechanotransduction), whereas hypoxic effects seem to play only a minor role. In the context of OTM, the hypoxic marker HIF-1α does not appear to be primarily stabilized by a reduced O₂supply but is rather stabilised mechanically.

During orthodontic tooth movement (OTM) to therapeutically correct the position of misaligned teeth, thus improving oral health and quality of life, fibroblasts, macrophages, and other immune cells within the periodontal ligament (PDL), which connects a tooth to its surrounding bone, are exposed to compressive and tensile strain. While it is known that PDL fibroblasts are critically involved in the biological regulation of OTM by a mechanotransductively triggered release of cytokines, it is unclear whether macrophages also react to pressure and tension in a similar manner thus impacting on or mediating OTM. RAW264.7 macrophages were seeded onto conventional 6-well cell culture plates for pressure or on Bioflex plates for tension assays and preincubated for 24 h. Forin vitrosimulation of physiological orthodontic compressive or tensile strain for 2 h, 4 h, 24 h, and 48 h, glass discs (2 g/cm²) were placed or adherent macrophages isotropically stretched for 16%, respectively. We determined cell number, cytotoxicity, and gene/protein expression ofVegf-a/VEGF-A (macrophage-mediated angiogenesis),Mmp-8/9(extracellular matrix reorganization), andCox-2/PG-E2,Il-6/IL-6, andTnf-α/TNF-α(proinflammatory mediators) by RT-qPCR and ELISA. Compressive but not tensile strain resulted in a significant reduction in cell number after only 2 h.Mmp-8andMmp-9expression was significantly enhanced within 24 h of compressive and in part tensile strain. Significantly increasedVegf-a/VEGF-A expression was detected within 4 h of pressure, but not during application of tensile strain. Expression of proinflammatory mediatorsCox-2/PG-E2,Il-6/IL-6, andTnf-α/TNF-αwas significantly increased as early as 2-4 h after application of compressive or tensile strain. Our results indicate that macrophages respond early on to compressive and tensile strain occurring during OTM with an enhanced gene expression of proinflammatory cytokines, which could affect PDL fibroblasts, osteoblasts, and immune cells triggering or enhancing the biological mechanisms and osteoclastogenesis underlying OTM.