Induction of Osteoclast Differentiation with RANKL-releasing Microspheres

Physical injury to the bone, such as a fracture or surgical intervention induces acute acceleration of bone turnover, also known as regional acceleratory phenomenon. Small injuries induce widespread activation, a principle that is utilized in corticotomy, where multiple bur marks are surgically placed in the alveolar bone to accelerate orthodontic tooth movement. Objectives: To create and test molecular tools for inducing regional acceleratory phenomenon by focal delivery RANKL, the chief osteoclast-differentiating molecule to osteoclast precursors. Methods: RANKL or bovine serum albumin (BSA) as a test protein is encapsulated in alginate microspheres (2.5%w/v) with the external gelation method using a custom-made atomizer. Peptide delivery into the incubation medium from microspheres is monitored with Bradford assay using NanoDrop ND-1000 spectrophotometer. Mouse bone marrow cells are isolated from mouse femurs and cultured in the presence of macrophage colony-stimulating factor (M-CSF). RANKL-loaded alginate microspheres are added to bone marrow cultures and osteoclast differentiation is monitored using tartrate-resistant acid phosphatase (TRAP) staining and morphological analysis. Results: The test protein BSA is released from alginate microspheres in a dose-dependent manner in the range of 0.01-10mg protein in 200µL incubation medium. Time course studies with 0.1mg BSA-loaded microspheres showed a maximum of 72µg/mL BSA concentration in the culture medium at 3 hours of incubation. Chitosan coating of alginate microspheres prolonged release with maximum BSA concentration of 54µg/mL at 24 hours of incubation. Alginate microsphere-encapsulated RANKL (100µL microsphere slurry at 10µg/mL) induced the development of TRAP-positive and morphologically distinct multinuclear osteoclast-like cells in M-CSF-treated bone marrow cultures. Conclusion: Alginate microspheres are suitable for rapid focal delivery of RANKL to osteoclast precursors. Microsphere-mediated RANKL delivery may offer an alternative approach to surgically-induced acceleration of bone remodeling. Supported by NIH NIDCR R01 DE016933.