RANK/RANKL/OPG signaling pathways in necrotic jaw bone from bisphosphonate-treated subjects

Abstract

Osteonecrosis of the jaw (ONJ) is a chronic complication affecting long-term bisphosphonate-treated subjects, recognized by non-healing exposed bone in the maxillofacial region. The pathophysiological mechanism underlying ONJ has not been fully elucidated. The aim of the present study was to investigate the role of RANK/RANKL/OPG signaling pathway and, in parallel, to evaluate angiogenic and matrix mineralization processes in jaw bone necrotic samples obtained from bisphosphonate-treated subjects with established ONJ. Necrotic bone samples and native bone samples were processed for Light and Field Emission in Lens Scanning Electron Microscope (FEISEM) analyses, for Real-Time RT-PCR to evaluate the gene expression of TNFRSF11A (RANK), TNFSF11 (RANKL), and TNFSF11B (OPG) and for immunohistochemical analyses of VEGF and BSP expression. Morphological analyses performed by Light microscope and FEISEM show empty osteocytic lacunae and alteration of lamellar organization with degradation of the mineralized bone matrix in necrotic bone samples. A significant increase in TNFRSF11A, TNFSF11, TRAF6 and NFAT2 gene expression, and a reduction of TNFSF11B gene transcription level compared is also showed in necrotic bone compared to control samples. No significant difference of VEGF expression is evidenced, while lower BSP expression in necrotic bone compared to healthy samples is found. Even if the pathogenesis of bisphosphonate-associated ONJ remains unknown, a link between oral pathogens and its development seems to exist. We suppose lipopolysaccharide produced by bacteria colonizing and infecting necrotic bone and the surrounding viable area could trigger RANK/RANKL/OPG signaling pathway and, in this context, osteoclasts activation could be considered as a protective strategy carried out by the host bone tissue to delimitate the necrotic area and to counteract infection.

Figure 1.

Hematoxylin and eosin staining of native (A) and necrotic (B) bone samples. Arrows indicate empty lacunae in necrotic bone samples.

Figure 2.

FEISEM analysis of native (A, B) and necrotic (C, D) bone samples, at different magnification.

Figure 3.

Relative gene expression of TNFRSF11A, TNFSF11, TRAF6, NFAT2 and TNFRSF11B in jaw bone, as determined by real-time RT-PCR. Bar graph represents the means ± SEM of three independent experiments (**P<0.01, ***P<0.001 vs control). Y-axis, fold change.

Figure 4.

Immunohistochemical analysis of VEGF and BSP expression in native and necrotic bone samples. A) VEGF expression in bone tissue specimens obtained from healthy donors. B) VEGF expression in bone tissue specimens obtained from BP-treated donors. C) BSP expression in bone tissue specimens obtained from healthy donors. D) BSP expression in bone tissue specimens obtained from bisBP-treated donors. N(-) negative control. E) Graphic representation of VEGF and BSP positive area % (± SD) densitometric analysis determined by direct visual counting of ten fields (mean values) for each of five slides per specimen at 20x magnification; blue bars, native bone samples; red bars, necrotic bone samples; *P<0.05.