IL-1β and Compressive Forces Lead to a Significant Induction of RANKL-expression in Primary Human Cementoblasts

Aim: The aim of this study was to investigate the response of primary human cementoblasts to conditions as they occur on the pressure side during orthodontic tooth movement.

Methods: In our previous study, the cementoblasts were characterized using markers for osteoblastogenic differentiation and the cementoblast-specific marker CEMP-1. Initially, primary human cementoblasts were compressed for 1 h, 4 h, and 6 h (30 g/cm(2)). In the second experiment, the cementoblasts were stimulated with interleukin (IL)-1β for 24 h and for 96 h with 1 ng/ml and 10 ng/ml and subsequently compressed for 1 h and 6 h. Changes in mRNA expression for receptor activator of NF-κB (RANK), RANK ligand (RANKL), osteoprotegerin (OPG), and cyclooxygenase-2 (COX-2) were measured by quantitative real-time polymerase chain reaction (RT-PCR). RANK and RANKL were also examined by immunocytochemical staining at the protein level.

Results: Compression (30 g/cm(2)) led to a significant increase in RANKL expression after 6 h. OPG expression in compressed cementoblasts was significantly reduced after 1 h. RANK remained unchanged during the course of the experiment. Stimulation with IL-1β induced RANKL and OPG expression. However, IL-1β-dependent induction of RANKL was more prominent than the induction of OPG, leading to a (significant) increase in the RANKL/OPG ratios. The expression of RANK remained unchanged after 24 h of stimulation with IL-1β and decreased significantly after 96 h. Compression of the prestimulated cells resulted in a further increase in RANKL expression significant after 6 h. OPG and RANK expression remained unchanged compared to the unstimulated sample. COX-2 increased significantly after both compression and stimulation with IL-1β. Combined stimulation and compression resulted in a significant further increase after 6 h compared to IL-1β stimulation alone.

Conclusion: Primary human cementoblasts in vitro express increased levels of RANKL, in particular during the combination of inflammation and compression. The increase in RANKL expression is not compensated by an increase in OPG expression. The induction of RANKL expression was associated with a significant increase in COX-2 expression. Since RANKL attracts osteoclasts, its increase might be associated with the progression of root resorption. The in vitro alterations in cementoblasts we observed may be indicators of cellular mechanisms that lead to the increased root resorption during orthodontic treatment.