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

Abstract

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.

Role of Oxygen Supply in Macrophages in a Model of Simulated Orthodontic Tooth Movement

“…Damage-associated molecular patterns are released by necrotic cell death so that the inflammatory cascade can begin [ 28 ]. As an initial reaction, cytokines such as TNF and IL-6 are secreted [ 7 ]. Both inflammation markers are significantly involved in OTM but are not known classical HIF-1 α target genes [ 29 ].…”

“…Therefore, approximately 250,000 RAW264.7 macrophages per ml were seeded onto sterile 6-well cell culture plates (353046, Omnilab) and cultivated for 24 h before the start of the experiment. Compressive force application was performed by using glass plates with a defined weight of 2 g/cm 2 for additional 24 h under normoxic or hypoxic conditions according to an established and published model [ 7 , 16 , 17 ] ( Figure 1 ). Hypoxic conditions were achieved by using a GasPak EZ gas development bag system with an indicator (260683, BD Biosciences) according to the manufacturer's instructions increasing CO 2 to at least 10% within 24 h. Therefore, we also measured pH (Seven Easy in combination with Inlab Expert pro electrode, Mettler Toledo) and detected a slight, but significant, effect of CO 2 on pH in the cell culture media (Supplemental figure 1 ).…”

“…RT-qPCR amplification and quality control was performed as described before [ 7 , 17 , 20 – 22 ] with the Mastercycler® ep realplex-S Thermocycler (Eppendorf AG, Hamburg, Germany). We constructed all primers ( Table 1 ) according to MIQE quality guidelines and criteria as described before [ 21 ].…”

“…Approximately 250,000 RAW264.7 macrophages per ml were seeded either onto conventional polystyrene plates (353046, Omnilab) or on lumox plates. After 24 h of preincubation, macrophages were either compressed using glass plates with a defined weight of 2 g/cm 2 according to an established and published model [7,16,17] or left untreated for further 24 h (Figure 1). To correspond to the results from repeated Experiment 1 with the addition of DMOG, we also repeated this experiment with compressive force treatment for 4 h (Supplemental figures 8-11).…”

“…It has been reported that a compression of blood vessels within the periodontal ligament occurs during orthodontic tooth movement (OTM) leading to a decreased local perfusion and concomitant reduction in oxygen supply (hypoxia) [ 2 ]. Cytokines and other inflammatory markers are secreted into the periodontal tissue by periodontal fibroblasts [ 3 , 4 ] or immune cells [ 5 ] to attract additional leukocytes and macrophages [ 6 , 7 ] inducing a “pseudoinflammatory process” [ 1 ]. This process is characterized by a promotion of inflammation but may also accelerate other noninflammatory processes mediated by periodontal ligament cells [ 1 ].…”