99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin

Abstract

The aim of the present study was to examine the influence of technetium methylenediphosphonate (99Tc - MDP) on the proliferation and differentiation of human osteoblasts. Human iliac cancellous bone was isolated and cultured with either 99Tc-MDP, β fibroblast growth factor (as a positive control) or medium only (as a negative control). Proliferation was assessed by direct cell counting, CCK-8 assay and bromo-deoxyuridine staining. The cell cycle and rate of apoptosis was assessed by propidium iodide staining and flow cytometry. Alkaline phosphatase (ALP) activity was assessed by the p-nitrophenyl phosphate method and mineralized nodules were stained with Alizarin Red. Expression of osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2) was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and expression levels of osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) were assessed by RT-qPCR and ELISA. Isolated human osteo-blasts stained positively for ALP and developed mineralized nodules. Treatment with 10-5-10 -10 M 99Tc-MDPenhanced proliferation and 48h incubation with 10-8 M 99Tc - M D P increased the proportion of cells in S-phase, decreased the proportion in G0/G1 phase, and increased the cell proliferation index. The rate of apoptosis also increased, but the increase was not sign ifica nt. Cells incubated with 10-6-10-9 M 99Tc-MDP for 3-9 days exhibited increased ALP activity and mineralized nodule development. 10-8 M 99Tc-MDP increased BMP-2 and OPG expression levels and OPG secretion, but OCN mRNA expression levels and RANKL secretion were not significantly altered at 72 h. 99Tc-MDP treatment induced osteoblast proliferation and differentiation without affecting apoptosis. These findings provide proof of concept for the future use of 99Tc-MDP in the treatment of bone-destructive diseases.