Effect of compressive loading and incubation with clodronate on the RANKL/OPG system of human osteoblasts

Abstract

Objectives: In vivo studies have shown that bisphosphonates result in slow rates of orthodontic tooth movement. This study investigated whether clodronate modifies the impact of mechanical loading on the RANKL/OPG system of human osteoblasts.

Methods: Osteoblasts were cultured in vitro with 0.5 or 5.0 µM clodronate for 48 h and/or subjected to 3 h of compressive loading at 34.9 g/cm(2). Cell viability was determined by MTT assay. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemical staining were used to analyze the cells for their production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) at the transcriptional and protein levels.

Results: Compressive loading did not affect osteoblast viability in a significant way. Clodronate (5.0 µM) mildly reduced the viability of both compressed and uncompressed cells. Compressive loading induced a 4.2-fold increase in RANKL gene expression, while clodronate led to a concentration-dependent inhibition of this effect (1.8-fold increase at 5.0 µM). OPG gene expression was decreased by compressive loading both in the presence of 0.5 µM clodronate and in the absence of clodronate, and OPG protein synthesis in the compressed cells was significantly decreased in the presence of clodronate. Immunocytochemical staining revealed an increase of RANKL protein synthesis in compressed cells, while clodronate and cell compression reduced this increase.

Conclusion: This study demonstrates that clodronate decreases the compression-induced RANKL/OPG ratio expressed by human osteoblasts. Reported in vivo findings of reduced osteoclast numbers on the compression side of orthodontic tooth movement under the action of clodronate-and the associated slow rate of tooth movement-might be attributable not only to a direct impact on osteoclasts but also to changes in osteoblast-osteoclast interaction resulting from the presence of clodronate.

Keywords: Bisphosphonates; Mechanical loading; Orthodontic tooth movement; RANKL/OPG system; Root resorption.