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.

Selection of appropriate housekeeping genes is essential for the validity of data normalization in reverse transcription quantitative PCR (RT-qPCR). Synovial fibroblasts (SF) play a mediating role in the development and progression of osteoarthritis (OA) pathogenesis, but there is no information on reliable housekeeping genes available. Therefore the goal of this study was to identify a set of reliable housekeeping genes suitable for studies of mechanical loading on SF from healthy and OA patients. Nine genes were evaluated towards expression stability and ranked according their relative stability determined by four different mathematical procedures (geNorm, NormFinder, BestKeeper and comparative ΔC_q). We observed that RPLP0 (ribosomal protein, large, P0) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) turned out to be the genes with the most stable expression in SF from non-OA or OA patients treated with or without mechanical loading. According to geNorm two genes are sufficient for normalization throughout. Expression of one tested target gene varied considerably, if normalized to different candidate housekeeping genes. Our study provides a tool for accurate and valid housekeeping gene selection in gene expression experiments on SF from healthy and OA patients with and without mechanical loading in consistent with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and additionally demonstrates the impact of proper housekeeping gene selection on the expression of the gene of interest.

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.

Genetic predisposition, traumatic events, or excessive mechanical exposure provoke arthritic changes in the temporomandibular joint (TMJ). We analysed the impact of mechanical stress that might be involved in the development and progression of TMJ osteoarthritis (OA) on murine synovial fibroblasts (SFs) of temporomandibular origin. SFs were subjected to different protocols of mechanical stress, either to a high-frequency tensile strain for 4 h or to a tensile strain of varying magnitude for 48 h. The TMJ OA induction was evaluated based on the gene and protein secretion of inflammatory factors (Icam-1, Cxcl-1, Cxcl-2, Il-1ß, Il-1ra, Il-6, Ptgs-2, PG-E2), subchondral bone remodelling (Rankl, Opg), and extracellular matrix components (Col1a2, Has-1, collagen and hyaluronic acid deposition) using RT-qPCR, ELISA, and HPLC. A short high-frequency tensile strain had only minor effects on inflammatory factors and no effects on the subchondral bone remodelling induction or matrix constituent production. A prolonged tensile strain of moderate and advanced magnitude increased the expression of inflammatory factors. An advanced tensile strain enhanced the Ptgs-2 and PG-E2 expression, while the expression of further inflammatory factors were decreased. The tensile strain protocols had no effects on the RANKL/OPG expression, while the advanced tensile strain significantly reduced the deposition of matrix constituent contents of collagen and hyaluronic acid. The data indicates that the application of prolonged advanced mechanical stress on SFs promote PG-E2 protein secretion, while the deposition of extracellular matrix components is decreased.

Dietary salt uptake and inflammation promote sodium accumulation in tissues, thereby modulating cells like macrophages and fibroblasts. Previous studies showed salt effects on periodontal ligament fibroblasts and on bone metabolism by expression of nuclear factor of activated T-cells-5 (NFAT-5). Here, we investigated the impact of salt and NFAT-5 on osteoclast activity and orthodontic tooth movement (OTM). After treatment of osteoclasts without (NS) or with additional salt (HS), we analyzed gene expression and the release of tartrate-resistant acid phosphatase and calcium phosphate resorption. We kept wild-type mice and mice lacking NFAT-5 in myeloid cells either on a low, normal or high salt diet and inserted an elastic band between the first and second molar to induce OTM. We analyzed the expression of genes involved in bone metabolism, periodontal bone loss, OTM and bone density. Osteoclast activity was increased upon HS treatment. HS promoted periodontal bone loss and OTM and was associated with reduced bone density. Deletion of NFAT-5 led to increased osteoclast activity with NS, whereas we detected impaired OTM in mice. Dietary salt uptake seems to accelerate OTM and induce periodontal bone loss due to reduced bone density, which may be attributed to enhanced osteoclast activity. NFAT-5 influences this reaction to HS, as we detected impaired OTM and osteoclast activity upon deletion.