TNF-α-induced bone resorption model using CHP nanogel in mice

Objectives: TNF-α is a pivotal cytokine in lipopolysaccharide (LPS)-induced bone resorption. We have previously found that LPS-induced bone resorption is significantly increase in TNF receptor type 2 (TNFR2)-deficient mice compared to the wild type mice. It is, however, difficult to clarify the mechanism of bone resorption induced by LPS since many cytokines are secreted after LPS injection. Therefore we tried to establish a simple bone resorption model using TNF-α. Nanogel of cholesterol bearing pullulan (CHP) have developed as nanocarrier which can form complex with hydrophobic drugs or protein for the controlrelease. In this study, we tested whether the CHP-TNF-α complex could induce bone resorption or notand tried to establish the TNF-α-induced bone resorption model. Methods: Five-week-old male TNFR2-deficient mice were used. Either 2 μg of TNF-α in 50 μl PBS or TNF-α/CHP solution was injected once a day for 4 days subcutaneously onto the calvaria of each mice and sacrificed 24h after the last injection. Calvariae were dissected and fixed for 3 days. Then they were subjected to μCT analysis and to the bone mineral density (BMD) measurement using dual x-ray absorptiometry. Results: 3D-μCT reconstruction images showed many resorption pits on calvariae in the TNF-α/CHP injected group, but not in the TNF-α injected group. The calvarial BMD in the TNF-α/CHP injected group was significantly decreased compared to the vehicle/CHP injected group (12.2 ± 0.1 vs 11.27 ± 0.55 (mg/cm², p<0.05), but not in the TNF-α injected group vs vehicle injected group. (11.9 ± 0.4 vs 12.4 ± 0.5 (mg/cm², p=0.23) Conclusion: The bone resorption model by using CHP nanogel might be suitable for evaluating the TNF-α-induced bone resorption.