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.

Myeloid cells regulate bone density in response to increased salt (NaCl) intake via the osmoprotective transcription factor, nuclear factor of activated T cells‐5 (NFAT‐5). Because orthodontic tooth movement (OTM) is a pseudoinflammatory immunological process, we investigated the influence of NaCl and NFAT‐5 on the expression pattern of macrophages in a model of simulated OTM. RAW264.7 macrophages were exposed for 4 h to 2 g cm−2 compressive or 16% tensile or no mechanical strain (control), with or without the addition of 40 mm NaCl. We analyzed the expression of inflammatory genes and proteins [tumor necrosis factor (TNF), interleukin (IL)‐6 and prostaglandin endoperoxide synthase‐2 (Ptgs‐2)/prostaglandin E2 (PG‐E2)] by real‐time‐quantitative PCR and ELISA. To investigate the role of NFAT‐5 in these responses, NFAT‐5 was both constitutively expressed and silenced. Salt and compressive strain, but not tensile strain increased the expression of NFAT‐5 and most tested inflammatory factors in macrophages. NaCl induced the expression of Ptgs‐2/PG‐E2 and TNF, whereas secretion of IL‐6 was inhibited. Similarly, a constitutive expression of NFAT‐5 reduced IL‐6 expression, while increasing Ptgs‐2/PG‐E2 and TNF expression. Silencing of NFAT‐5 upregulated IL‐6 and reduced Ptgs‐2/PG‐E2 and TNF expression. Salt had an impact on the expression profile of macrophages as a reaction to compressive and tensile strain that occur during OTM. This was mediated via NFAT‐5, which surprisingly also seems to play a regulatory role in mechanotransduction of compressive strain. Sodium accumulation in the periodontal ligament caused by dietary salt consumption might propagate local osteoclastogenesis via increased local inflammation and thus OTM velocity, but possibly also entail side effects such as dental root resorptions or periodontal bone loss.