Co-culture of osteoblastic and endothelial cells in the presence of bisphosphonates

Introduction. Long-term therapies with Bisphosphonates (BPs) can cause osteonecrosis of the jaws, a condition characterized by tissue dehiscence, chronic bone devitalization, hypocellularity and lytic radiographic features, being usually refractory to therapy. Among the diverse and complex mechanisms of action recently suggested for BPs, the anti-angiogenic properties appears to be a relevant feature of their pharmacological profile. Due to the intimate relationship between angiogenesis and osteogenesis during bone formation events, the aim of this study was to analyze the effect of representative BPs – alendronate and zoledronate, two widely used BPs in therapeutics - in the behavior of a co-culture system of human osteoblastic and endothelial cells.

Methods. MG63 osteoblast-like cells (10³ cell/cm²) and human dermal microvascular endothelial cells (10⁴ cell/cm²) were cultured isolated or co-cultured in endothelium medium, in the presence of 10^-12 to 10^-6 M Alendronate or Zolendronate. Cultures were maintained for 14 days and characterized for cell viability/proliferation (MTT assay), pattern of cell growth (CLSM) and gene expression of osteoblastic and endothelial markers (RT-PCR; Collagen type1, ALP, BMP-2, OPG, M-CSF, CD31, VE-Cadherin and vWF).

Results. In control conditions (absence of BPs), co-cultures of osteoblast and endothelial cells maintained the viability/proliferation and presented a characteristic pattern of cell growth, i.e. the formation of cell clusters of endothelial cells surrounded by osteoblast cells, and inductium and/or earlier expression of osteoblastic and endothelial markers. The presence of Alendronate or Zolendronate did not affect cell viability/proliferation but caused decreased gene expression of endothelial associated markers in monocultures and co-cultures.

Conclusion. The inhibitory effects of BPs in endothelial cells might play a role in the deleterious effects of BPs in the bone tissue.