W9 peptide (W9) has been proven to inhibit inflammatory bone resorption by blocking both tumor necrosis factor (TNF)-α and receptor activator of NF-κB ligand (RANKL) signaling. Recently, we found this bone resorption inhibitor promoted BMP-2 induced bone formation. Given that TNF-α is proven to be an inhibitor of bone formation, we hypothesized the synergistic effects of W9 on bone formation could be TNF-α-dependent. The present study was undertaken to clarify whether the stimulatory effects of W9 on bone formation is TNF-α dependent or not.
Methods:
Ectopic bone formation model was used in this study. Collagen, containing either BMP-2 (1 μg) alone or BMP-2 (1 μg) with W9 (0.56 mg ), was implanted into the five-week-old WT, TNF-α-deficient, TNFR1-deficient, and RANKL-deficient mice back muscle. They were sacrificed at 12 days after implantation. Radiological and histological analyses were performed.
Results:
We confirmed that W9 promoted BMP-2-induced ectopic bone formation. Surprisingly, μ-CT images showed that W9 also promoted ectopic bone formation in both TNFR1-deficient and TNF-α-deficient mice. These findings were confirmed by the quantitative analysis by using dual-energy-X-ray-absorptiometry. In WT mice, W9 increased bone mineral content (BMC) of ectopic bone by 2.4 fold more compare to the ectopic bone induced by BMP-2 alone. W9 also increased BMC of ectopic bone induced by BMP-2 in both TNFR1-deficient and TNF-α deficient mice to the same extent as in WT mice (by 3.3-fold and 2.2-fold, respectively). Therefore, we performed the same experiment using RANKL-deficient mice as W9 is shown as a RANKL-antagonist as well as a TNF-α-antagonist. The acceleration of ectopic bone formation by W9 was blunted in RANKL-deficient-mice.
Conclusions:
Although further studies are necessary to clarify the RANKL-dependent mechanism, we speculated that the stimulatory effects of W9 on bone formation might be regulated by RANKL-related signaling not TNF-α.