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. For in vitro simulation 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 of Vegf-a/VEGF-A (macrophage-mediated angiogenesis), Mmp-8/9 (extracellular matrix reorganization), and Cox-2/PG-E2, Il-6/IL-6, and Tnf-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-8 and Mmp-9 expression was significantly enhanced within 24 h of compressive and in part tensile strain. Significantly increased Vegf-a/VEGF-A expression was detected within 4 h of pressure, but not during application of tensile strain. Expression of proinflammatory mediators Cox-2/PG-E2, Il-6/IL-6, and Tnf-α/TNF-α was 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.

Summary Background/objective Periodontal ligament fibroblasts (PDLF) play an important mediating role in orthodontic tooth movement expressing various cytokines, when exposed to compressive or tensile strain. Here, we present a simplified and easy-to-handle, but reliable and valid method for simulating static isotropic tensile strain in vitro using spherical silicone cap stamps. Furthermore, we identify appropriate reference genes for data normalization in real-time quantitative polymerase chain reaction (RT-qPCR) experiments on PDLF subjected to tensile strain. Materials and methods PDLF were cultivated on flexible bioflex membranes and exposed to static isotropic tensile strain of different magnitudes and timeframes. We determined cell number, cytotoxicity, and relative expression of proinflammatory genes cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). For normalization of RT-qPCR data, we tested the stability and validity of nine candidate reference genes with four mathematical algorithms (geNorm, NormFinder, comparative ΔCq, and BestKeeper) and ranked them based on their calculated expression stability. Results We observed no decrease in cell number or cytotoxic effect at any of the applied magnitudes and timeframes of tensile strain. At 16 per cent and 35 per cent tensile strain for 48 hours, we detected a significant increase in COX-2 and decrease in IL-6 gene expression. Highest stability was found for TBP (TATA-box-binding protein) and PPIB (peptidylprolyl isomerase A) in reference gene validation. According to the geNorm algorithm, both genes in conjunction are sufficient for normalization. In contrast to all other candidate genes tested, gene expression normalization of target gene COX-2 to reference genes EEF1A1, RPL22, and RNA18S5 indicated no significant upregulation of COX-2 expression. Conclusions A strain magnitude of 16 per cent for 48 hours elicited the most distinct cellular response by PDLF subjected to static tensile isotropic strain by the presented method. TBP and PPIB in conjunction proved to be the most appropriate reference genes to normalize target gene expression in RT-qPCR studies on PDLF subjected to tensile strain.

Osteoarthritis (OA) affects the integrity of the entire joint including the synovium. The most abundant cells in the synovium are fibroblasts (SF). Excessive mechanical loading might contribute to OA pathogenesis. Here, we investigate the effects of mechanical loading on SF derived from non-OA (N-SF) and OA patients (OA-SF). We treated N-SF and OA-SF with or without mechanical loading for 48h after 24h of preincubation. Then we assessed gene and protein expression of proinflammatory factors (TNFα, COX-2, PG-E2, IL-6), extracellular matrix (ECM) components (COL1, FN1) and glycosaminoglycans (GAGs) via RT-qPCR, ELISA, DMMB assay and HPLC. Mechanical loading significantly increased TNFα and PG-E2 secretion by N-SF and OA-SF, whereas in OA-SF IL-6 secretion was reduced. COL1 and FN1 secretion were downregulated in N-SF during loading. OA-SF secreted less COL1 compared to N-SF under control conditions. In contrast, OA-SF in general expressed more FN1. GAG synthesis was upregulated in N-SF, but not in OA-SF during loading with OA-SF displaying a higher charge density than N-SF. Mechanical loading enhanced proinflammatory factor expression and GAG synthesis and decreased secretion of ECM components in N-SFs, indicating a contributing role of SF to OA development.

Increased salt (NaCl) consumption triggers chronic diseases such as hypertension or osteopenia. Its impact on orthodontic tooth movement and periodontitis, however, has not been investigated, although both processes are related to the immune system, with periodontal ligament fibroblasts (PDLFs) playing a key mediating role. Here, we investigated the impact of NaCl on the expression pattern of PDLFs in a model of simulated compressive orthodontic strain. Periodontal ligament fibroblasts were preincubated for 24 h with additional 0 or 40 mM NaCl and concurrently treated for another 48 h with or without compressive strain of 2 g cm−2. We analyzed the expression of genes and proteins involved in orthodontic tooth movement by reverse transcription quantitative polymerase chain reaction (RT‐qPCR), ELISA, and immunoblot. Co‐culture experiments were performed to observe PDLF‐mediated osteoclastogenesis. A higher (40 mM) concentration of NaCl in the culture medium resulted in increased secretion of prostaglandin, expression of alkaline phosphatase, and expression of genes involved in extracellular matrix remodeling, but decreased compression‐induced expression of the interleukin‐6 (IL6) gene. The 40 mM concentration of NaCl also enhanced receptor activator of nuclear factor kappa‐B ligand (RANKL) but reduced that of osteoprotegerin (OPG), resulting in upregulated PDLF‐mediated osteoclastogenesis. A high NaCl concentration in the periodontal ligament, corresponding to a high‐salt diet in vivo, may influence orthodontic tooth movement and periodontitis through increased secretion of prostaglandins by PDLFs and upregulated PDLF‐mediated osteoclastogenesis, possibly accelerating orthodontic tooth movement and propagating periodontitis and periodontal bone loss.

Background This study aimed to investigate, if different physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fibroblasts induced by simulated orthodontic compressive strain. Methods A pool of hPDL fibroblasts was treated in absence or presence of 25(OH)D3 in 3 different concentrations (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fibroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxylase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non-parametric tests were used with an alpha of 5%. Results RANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were differentially expressed during simulated orthodontic compressive strain (p < 0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean = 0.96 ± 0.68, mean = 1.61 ± 0.66 and mean = 1.86 ± 0.78, respectively) in comparison to the control (mean 2.58 ± 1.16) (p < 0.05). CYP2R1 gene expression was statistically modulated by the different 25(OH)D3 concentrations applied (p = 0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p < 0.05). Conclusions Simulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regulate the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fibroblasts in the context of orthodontic compressive strain. Our study also suggests that single nucleotide polymorphisms in the VDR gene regulate VDR expression in periodontal ligament fibroblasts in the context of orthodontic compressive strain.