Pentamidine Inhibits Titanium Particle-Induced Osteolysis In Vivo and Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation In Vitro

Abstract

Background: Wear debris-induced osteolysis leads to periprosthetic loosening and subsequent prosthetic failure. Since excessive osteoclast formation is closely implicated in periprosthetic osteolysis, identification of agents to suppress osteoclast formation and/or function is crucial for the treatment and prevention of wear particle-induced bone destruction. In this study, we examined the potential effect of pentamidine treatment on titanium (Ti) particle-induced osteolysis, and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis.

Methods: The effect of pentamidine treatment on bone destruction was examined in Ti particle-induced osteolysis mouse model. Ti particles were implanted onto mouse calvaria, and vehicle or pentamidine was administered for 10 days. Then, calvarial bone tissue was analyzed using micro-computed tomography and histology. We performed in vitro osteoclastogenesis assay using bone marrow-derived macrophages (BMMs) to determine the effect of pentamidine on osteoclast formation. BMMs were treated with 20 ng/mL RANKL and 10 ng/mL macrophage colony-stimulating factor in the presence or absence of pentamidine. Osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining, real-time polymerase chain reaction, and immunofluorescence staining.

Results: Pentamidine administration decreased Ti particle-induced osteoclast formation significantly and prevented bone destruction compared to the Ti particle group in vivo. Pentamidine also suppressed RANKL-induced osteoclast differentiation and actin ring formation markedly, and inhibited the expression of nuclear factor of activated T cell c1 and osteoclast-specific genes in vitro. Additionally, pentamidine also attenuated RANKL-mediated phosphorylation of IκBα in BMMs.

Conclusion: These results indicate that pentamidine is effective in inhibiting osteoclast formation and significantly attenuates wear debris-induced bone loss in mice.

Keywords: Osteoclastogenesis; Osteolysis; Pentamidine; RANKL; Titanium.

Fig. 1

Pentamidine prevented bone erosion in Ti particle-induced osteolysis in a mouse calvarial model. Ti particles were embedded on to the mouse calvaria, and injected with pentamidine (1 or 5 mg/kg) or vehicle control for 10 days. At the end of the study period, calvaria were dissected, fixed, and scanned with micro-CT. A Representative micro-CT images of calvaria from each group (n = 5). Scale bar = 5 mm. B Quantification of bone mineral density (BMD) and bone volume to tissue volume (BV/TV) ratio. **p < 0.01 and *p < 0.05. Pent: pentamidine

Fig. 2

Histological analysis of murine calvarial bone sections. A Representative images of hematoxylin and eosin (H&E), and tartrate-resistant acid phosphatase (TRAP) stained calvarial bone sections. Scale bar = 100 μm. B The number of TRAP-positive cells (yellow arrow head) per bone surface in each group (n = 5) was assessed (lower graph). *p < 0.05 versus Ti group. Pent: pentamidine

Fig. 3

Pentamidine suppressed RANKL-induced osteoclast differentiation in vitro and the expression of osteoclast-specific genes. A Bone marrow-derived macrophages (BMMs) were cultured in osteoclast-inducing medium containing M-CSF (10 ng/mL) and RANKL (20 ng/mL) in the presence or absence of various concentrations of pentamidine. After 4 days of culture, cells were fixed and stained for TRAP. Scale bar = 500 μm. B The number of TRAP-positive multinucleated cells was counted. C BMMs were treated with different doses of pentamidine in the presence of M-CSF for 3 days. Cell viability was determined by using MTT assay. D BMMs were incubated with M-CSF (10 ng/mL) and RANKL (20 ng/mL) in the presence or absence of 5 μM pentamidine for 4 days and mRNA levels of NFATc1 (Nfatc1), Cathepsin K (Ctsk), DC-STAMP (Dcstamp), and TRAP (Acp5) were determined by real-time PCR. **p < 0.01 and *p < 0.05 versus vehicle-treated control group

Fig. 4

Pentamidine inhibited actin ring formation and nuclear localization of NFATc1, and impaired RANKL-induced NF-κB signaling. A BMMs were seeded on glass coverslips and stimulated with M-CSF (10 ng/mL) and RANKL (20 ng/mL) in the presence or absence of 5 μM pentamidine. Nuclear localization of NFATc1 was examined by immunostaining. Nuclei and F-actin were labeled with DAPI and rhodamine-conjugated phalloidin, respectively. Scale bar = 50 μm. B Serum-starved BMMs were pretreated with or without 5 μM pentamidine for 1 h followed by stimulation with RANKL (50 ng/mL). Western blot analysis was performed to determine the levels of phosphorylated p38 (p-p38), p-ERK, p-JNK, and p-IκBα by using specific antibodies