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 bromodeoxyuridine 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 osteoblasts stained positively for ALP and developed mineralized nodules. Treatment with 10‑5‑10‑10 M 99Tc‑MDP enhanced proliferation and 48 h incubation with 10‑8 M 99Tc‑MDP 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 significant. 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.

Figure 1.

Morphology of passage 2 human osteoblasts. (A) Light microscopic image of human osteoblasts at day 3 (x100 magnification). (B) Image of human osteoblasts at day 6 (x100 magnification) and (C) Human osteoblasts at day 12 at 100% confluence (x40 magnification).

Figure 2.

Detection of osteoblast proliferation using bromodeoxyuridine staining 72 h following treatment (x200 magnification). (A) control, (B) β fibroblast growth factor and (C) technetium methylenediphosphonate.

Figure 3.

Influence of ⁹⁹Tc-MDP on the osteoblast cell cycle 48 h following treatment. (A): ⁹⁹Tc-MDP, (B): β fibroblast growth factor and (C): control. ⁹⁹Tc-MDP, technetium methylenediphosphonate.

Figure 4.

Evaluating the influence of ⁹⁹Tc-MDP (10⁻⁸ M) on osteoblast apoptosis using propidium iodide staining, detected by flow cytometry 48 h following treatment. Data are presented as the mean + standard deviation; n=3; P>0.05 vs. control. ⁹⁹Tc-MDP, technetium methylenediphosphonate; βFGF, β fibroblast growth factor.

Figure 5.

Influence of ⁹⁹Tc-MDP (10⁻⁸ M) on mineralized nodule formation. (A) Representative images of mineralized nodules, stained with Alizarin Red 14 days following treatment. (B). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. control. ⁹⁹Tc-MDP, technetium methylenediphosphonate; βFGF, β fibroblast growth factor.

Figure 6.

mRNA expression levels of (A) OCN, (B) BMP-2 and (C) OPG/RANKL in osteoblasts treated with 10⁻⁸ M ⁹⁹Tc-MDP, measured by reverse transcription-quantitative polymerase chain reaction following 72 h incubation. Data are expressed as the mean + standard deviation; n=3; *P<0.05 vs. control. OCN, osteocalcin; BMP-2, bone morphogenetic protein-2; OPG/RANKL, osteoprotegerin/receptor activator of NF-κB ligand; ⁹⁹Tc-MDP, technetium methylenediphosphonate; βFGF, β fibroblast growth factor.