Method: Five-week-old male TNF-type1-receptor (TNFR1) deficient, TNF-α deficient mice, receptor activator of NF-κB ligand (RANKL) deficient mice and the wild type (WT) mice were used. Collagen discs containing either BMP-2 (1 μg) alone or BMP-2 (1 μg) with 0.56 mg W9 were freeze-dried, and were implanted into the mice back muscle. Mice were sacrificed on day 12 after implant and radiological and histological analyses were performed.
Result: Micro-CT images showed that larger size (3-times bone mineral content) of ectopic bone was observed in BMP-2 with W9 group compared to BMP-2 alone in WT mice. Surprisingly, W9 also increased ectopic bone formation in both TNFR1 deficient and TNF-α deficient mice to the same extent as in WT mice. Since W9 is proven to bind to RANKL as well as TNF-α, we speculated that the stimulatory effects of W9 on BMP-2 induced bone formation might be regulated by the other mechanism such as RANKL-related signaling. Therefore, we performed the same experiment using RANKL deficient mice. As expected, the acceleration of ectopic bone formation by W9 was much less when compared to the WT type mice.
Conclusion:
These results suggest that TNF-α antagonist peptide W9, promotes BMP-2-induced bone formation at least partially due to the RANKL-dependent mechanism.
This study was collaborated with Nobuyuki Udagawa (Matsumoto Dental University) and Hisataka Yasuda (Oriental Yeast Ltd Co, Japan)