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

Abstract

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. 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. 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. 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.