Abstract
Background: Bisphosphonate related osteonecrosis of the jaw (BRONJ) is a widely recognised dental side effect of bisphosphonate therapy in which the key finding is a non-healing exposed necrotic bone in the oral cavity. The mechanisms underlying BRONJ are poorly understood. Evidence from previous studies indicates that inhibition of bone remodelling, toxic effects on soft tissues, anti-angiogenic effects, and the inhibition of the mevalonate pathway (MVP) may play a role in BRONJ pathogenesis.
Aims: To determine the effects of zoledronic acid (ZA) and replenishment of the MVP by geranylgeraniol (GGOH) on HGFs and HOBs.
Methods: HGFs were cultured from gingival tissue (n=5) and HOBs were isolated from mandibular alveolar bone (n=3). HGFs were phenotyped using vimentin staining and HOBs were phenotyped using an in vitro bone nodule formation assay, immunofluorescence for osteocalcin and staining for alkaline phosphatase, alizarin red and von kossa. Cellular behaviour was examined using a CellTiter-Blue® viability assay, IBIDI culture-Insert migration assay (HOBs only), and an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay. Cellular ultra-structure was examined by transmission electron microscopy (TEM). For HGFs, qRT2-PCR technology was used to examine gene expression profiles for VEGFA, BMP2, RHOB, EREG and IFNA1 with ELISAs for proteins levels of VEGFA and BMP2. Human osteogenic and angiogenic/anti-angiogenic PCR arrays (SABiosciences) were used for HOBs.
Results: ZA caused an initial increase in cell viability followed by a rapid dose-dependent decrease at each time point. The simultaneous addition of ZA and GGOH (for 72 hrs) partially restored cell viability. Caspase 3/7 was detected in ZA treated HGFs and HOBs indicating apoptosis. TEM revealed signs of apoptosis in ZA treated cells and the appearance of multiple lipid-like vesicles with the addition of GGOH coincided with cellular recovery processes. In HGFs ZA significantly (p ≤ 0.05, FR > ±2) up-regulated VEGFA, RHOB, BMP2 and EREG gene at one or more time points but not IFNA1. Addition of GGOH resulted in a reduction in the expression of all genes. The protein concentration of VEGFA was higher in ZA treated HGFs, however BMP2 proteins were not detected due to their rapid degradation. For HOBs, among the 168 osteogenic and angiogenic genes tested, 28 genes in each array were significantly up- or down-regulated (p ≤ 0.05, FR > ±2) in the presence of ZA compared with controls at one or more time points. The addition of GGOH caused a decrease in the expression of some genes.
Conclusions: ZA resulted in a decrease in cell viability, and migration (HOBs only), and induction of apoptosis. GGOH acted as a salvage pathway after ZA induced blockage of the MVP, and this was observed for the proliferation, apoptosis and migration assays and in some of the gene expression assays. The negative effects of ZA on HGFs and HOBs, and their reversal with the addition of GGOH, suggests that the effect of ZA on HGFs and HOBs is mediated via the MVP. The results also suggest a possible therapeutic/preventive strategy for BRONJ may be to target the MVP by reversing the effects of ZA with the addition of GGOH.