The role of mechanotransduction versus hypoxia during simulated orthodontic compressive strain—an in vitro study of human periodontal ligament fibroblasts

Abstract

During orthodontic tooth movement (OTM) mechanical forces trigger pseudo-inflammatory, osteoclastogenic and remodelling processes in the periodontal ligament (PDL) that are mediated by PDL fibroblasts via the expression of various signalling molecules. Thus far, it is unknown whether these processes are mainly induced by mechanical cellular deformation (mechanotransduction) or by concomitant hypoxic conditions via the compression of periodontal blood vessels. Human primary PDL fibroblasts were randomly seeded in conventional six-well cell culture plates with O2-impermeable polystyrene membranes and in special plates with gas-permeable membranes (Lumox®, Sarstedt), enabling the experimental separation of mechanotransducive and hypoxic effects that occur concomitantly during OTM. To simulate physiological orthodontic compressive forces, PDL fibroblasts were stimulated mechanically at 2 g·cm−2 for 48 h after 24 h of pre-incubation. We quantified the cell viability by MTT assay, gene expression by quantitative real-time polymerase chain reaction (RT-qPCR) and protein expression by western blot/enzyme-linked immunosorbent assays (ELISA). In addition, PDL-fibroblast-mediated osteoclastogenesis (TRAP+ cells) was measured in a 72-h coculture with RAW264.7 cells. The expression of HIF-1α, COX-2, PGE2, VEGF, COL1A2, collagen and ALPL, and the RANKL/OPG ratios at the mRNA/protein levels during PDL-fibroblast-mediated osteoclastogenesis were significantly elevated by mechanical loading irrespective of the oxygen supply, whereas hypoxic conditions had no significant additional effects. The cellular–molecular mediation of OTM by PDL fibroblasts via the expression of various signalling molecules is expected to be predominantly controlled by the application of force (mechanotransduction), whereas hypoxic effects seem to play only a minor role. In the context of OTM, the hypoxic marker HIF-1α does not appear to be primarily stabilized by a reduced O2 supply but is rather stabilised mechanically.

Transcriptional Expression in Human Periodontal Ligament Cells Subjected to Orthodontic Force: An RNA-Sequencing Study

“…Under tension force, PDL cells participate in DNA synthesis of osteoblasts; are differentiated to osteoblasts; or are released from G 2 block, which contributes to the alveolar bone formation. Moreover, morphological deformation of the PDL induced by mechanical force plays a key role in recovery of its original shape via the remodeling process [25]. Therefore, the upregulated cell cycle of the PDLs would make orthodontic tooth movement possible by facilitating the remodeling process.…”

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

“…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).…”

“…As these immune cells also constitute an important cell population within the periodontal ligament and are involved in the regulation and instigation of OTM at the cellular-molecular level [8], the aim of this work was therefore to clarify the relative impact of orthodontically induced mechanical strain and hypoxic conditions in the periodontal ligament on macrophages for the mediation and regulation of OTM focusing on HIF-1α expression and its stabilization as well as on genes and proteins involved in the inflammatory processes occurring during OTM. To address this question, we used an established in vitro model to simulate orthodontic force application and mechanical strain as well as hypoxic conditions [16][17][18].…”

“…In this work, we focus on the HIF 1 target genes vascular endothelial growth factor ( Vegf ) and cyclooxygenase-2 ( Cox-2 ). VEGF and COX-2 are important mediators for angiogenesis [ 14 ] and inflammation [ 15 ] and are upregulated in periodontal ligament cells during mechanical strain as occurring during OTM [ 4 , 16 , 17 ]. Cox-2 is increasingly expressed, particularly during mechanical strain and inflammation in order to catalyse the conversion of arachidonic acid to prostaglandins, most prominently prostaglandin E2 [ 16 , 18 ], which among others contributes to vasodilatation [ 11 ].…”

“…Fibroblasts of the periodontal ligament stabilized HIF-1 α protein after compressive force treatment. This stabilization was mainly due to mechanotransductive effects, whereas hypoxia played a minor role [ 17 ]. However, effects of compressive force treatment compared to oxygen supply on macrophages are so far unknown.…”