Bone Abstracts

Non-unions and critical size defects (CSD) are major orthopedic challenges. The gold standard for CSD treatment, bone grafting, is associated with several morbidities. Growth factors such as bone morphogenetic proteins (BMPs) have been utilized in clinic. Nevertheless, huge doses are required for therapeutic effects. Endogenous BMP antagonists, such as Noggin, could be inhibited using small interfering RNA (siRNA) to increase the bioavailability of BMPs and accelerate bone healing. Lipid-based nanoparticles (LNPs) are the most advanced delivery vehicles. We hypothesize that Noggin siRNA delivery by LNPs enhances osteogenesis and bone healing in CSD. We plan to test our hypothesis both in vitro and in vivo. We have characterized the physicochemical properties of an LNP and have assessed the ability of the LNP-siRNA complex to transfect cultured osteoblasts. Treatment with 50 nM fluorescently-labeled siRNA resulted in over 90% cell transfection and excellent cell viability, confirmed by FACS and confocal microscopy. Screening of a library of Noggin siRNAs revealed one siRNA with 60% Noggin knock-down after 24 h and 70% protein down-regulation after 48 h and a consequent 40% up-regulation in BMP downstream genes, Smad1 and 5. We observed a significant increase in both Alp activity and mineralized matrix formation after 10 days of LNP-siRNA treatment. in vivo effects of the LNP-Noggin siRNA system will be evaluated in a rat femoral CSD model. Preliminary results from serial in vivo imaging demonstrate localized accumulation of fluorescently labeled siRNA at the site of bone defect over time. Treated animals will be sacrificed at different time-points and the bone samples will be assayed by Western Blot, RT-qPCR and immunohistochemistry. The quality and quantity of newly formed bone will be evaluated by X-ray, μ-CT scan, histology, and biomechanical testing.

This study will provide a practical approach to accelerating bone formation via endogenous BMP signal up-regulation and enhancing the potency of minimal safe doses of exogenous BMP. The results from this study will pave the way for the safe clinical management of impaired bone healing by avoiding the administration of toxic doses of exogenous BMP.