Simplified method for applying static isotropic tensile strain in cell culture experiments with identification of valid RT-qPCR reference genes for PDL fibroblasts

Abstract

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.

Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts

“…Mechanical strain can induce inflammation and can alter extracellular matrix composition. In contrast, application of static tensile strain on fibroblasts of periodontal origin caused an anti-inflammatory reaction [21]. We saw an arthritis-associated inflammation in case of static isotropic tensile strain, as the proinflammatory markers (TNF-α, IL-1β, IL-6, COX-2) were significantly upregulated.…”

“…Gene expression analysis was performed using SYBR ® Green JumpStart™ Taq ReadyMix™ (S4438, Sigma-Aldrich, Steinheim, Germany) and 1 ng/µL cDNA sample as described before [21,26]. The sample (1.5 µL cDNA) was mixed with SYBR ® Green JumpStart™ Taq ReadyMix™ (7.5 µL), nuclease-free water (5.25 µL) and specific primers (0.375 µL, 10 pmol; primer synthesis and purification done by Eurofins MWG Operon LLC, Huntsville; High Purity Salt Free Purification HPSF ® ) on ice in 96-well PCR plates (TW-MT, 712282; Biozym Scientific GmbH, Hessisch Oldendorf, Germany), sealed for qPCR reaction (BZO Seal Filmcover; 712350; Biozym Scientific GmbH, Hessisch Oldendorf, Germany).…”

“…In general, in vitro experiments suffer from inadequate simulation of in vivo situations, especially when mimicking movement and the effect of mechanical strain on cells. For this reason, various protocols and applications are published aiming to overcome this issue [21][22][23][24].…”

Effects of Compressive and Tensile Strain on Macrophages during Simulated Orthodontic Tooth Movement

“…In parallel, on the same respective 6-well plates, RAW264.7 macrophages were cultivated for 2 h, 4 h, 24 h, and 48 h without compressive forces (control). 16% static isotropic tensile strain was applied to RAW264.7 macrophages under cell culture conditions at 70% confluency by means of spherical silicone stamps inserted from the well bottom into the flexible Bioflex membrane for 2 h, 4 h, 24 h, and 48 h according to an established and published method to simulate tensile orthodontic strain in in vitro experiments (Figure 1(b)) [20,21]. In parallel, RAW264.7 macrophages were again cultured on the same Bioflex 6-well plates without stretching for 2 h, 4 h, 24 h, and 48 h. RNA isolation was performed as described before [13].…”

“…Primers were synthesized and purified by Eurofins MWG Operon LLC (Huntsville, AL, USA; High Purity Salt Free Purification HPSF®) and were not modified ( Table 1). They were constructed using NCBI Primer-BLAST according to MIQE guidelines as described before [13,20,22]. For determination of relative gene expression, two reference genes in combination (EEF1A1 and SDHA) were used, which have been shown to be stably expressed in RAW264.7 macrophages under the conditions investigated (data not shown).…”