EFFECTS OF N-CONTAINING BISPHOSPHONATES ON HUMAN PRIMARY OSTEOBLASTS DERIVED FROM A HETEROGENEOUS PATIENT COHORT

Bisphosphonates (BPs) are prescribed to treat a wide range of skeletal disorders, including osteoporosis, Paget disease, osteogenesis imperfecta and primary or secondary bone cancer. Their pharmacological action is exerted through the accumulation in areas of active bone turnover and the direct inhibition of osteoclast-mediated bone resorption. Indeed, BPs induce osteoclast apoptosis by affecting essential pathways of cell metabolism: either forming non-functional ATP analogues (non-N-containing bisphosphonates) or interfering with mevalonate pathway (N-containing bisphosphonates). However, their role in bone formation and a possible direct effect on osteoblasts (OBs) is controversial and the determination of BP concentrations at different skeletal districts is still an open issue. A serious side effect of the treatment with BPs is the osteonecrosis of jaws (BRONJ), a necrotizing and progressive lesion affecting the mandibular and/or maxillary bone. This drug-related osteonecrosis, whose etiology is not completely unravelled yet, constitutes a severe odontostomatological problem that should not be underestimated. Here, I present the direct effects of two nitrogen containing bisphosphonates, Alendronate (AL) and Zoledronate (ZL), on primary human osteoblast viability, expression and secretion of ECM proteins, functional mediators and bone biomarkers. Osteoblasts, isolated from patients who underwent total hip replacement surgery at IRCCS Galeazzi Orthopaedic Institute, were incubated with several concentrations of AL and ZL (range from 10^-15 to 10^-5M). We never observed any peculiar effect on osteoblast vitality and proliferation, except a significant inhibition induced by both drugs at 10^-5M, indicating an in vitro cytotoxicity. ALP activity was minimally enhanced at day 14 by low BP doses, while higher concentrations slightly inhibited it. We then investigated the modulation of ECM protein expression after exposure to low, intermediate and high BP concentrations (10^-13, 10^-10 and 10^-7M): Osteonectin (SPARC) was increased at day 7 by both compounds, while type I Collagen and Osteopontin (OPN) were clearly down-modulated only by AL. The secretion of bone biomarkers and cytokines was also analysed in supernatants of cultured osteoblasts at day 3. No overall effect was observed on Osteoprotegerin (OPG), DKK1 and IL-6. The levels of released TNFα, Sclerostin (SOST) and IL-1β, all inhibitors of osteoblast differentiation, were slightly enhanced by both drugs, independently on their concentrations, while OPN was mildly reduced only by 10^-7M BPs. Interestingly, DKK-1, IL-6 and Osteocalcin (OC) secretion was increased in all tested conditions by cells derived from donors <50 years old, suggesting that this subpopulation might respond more rapidly to the treatments. In conclusion, my results indicate that AL and ZL in concentrations below µM do not overall impair osteoblast viability, nor most of the analysed biomarkers. However, since SPARC expression, SOST, TNFα and IL-1β secretion are increased, we believe that accumulated doses of these BPs may alter bone turnover either through a direct action on bone-forming cells or by influencing the release of mediators involved in cell-cell communication. Despite the heterogeneity of our samples, I am confident to suggest that the variable in vitro effect of BPs on OBs derived from either young or elderly donors might mediate a different therapeutic action or an increased risk of developing collateral effects.