Novel Oxysterol Induces Osteogenic Differentiation in Rabbit Bone Marrow Stem Cells

Purpose: Bone morphogenetic proteins (BMPs) have played a central role in the development of regenerative therapies for bone reconstruction, however, they come at a high cost and side effect profile. Oxysterols are naturally occurring and inexpensive oxidation products of cholesterol that can induce osteogenic differentiation in mesenchymal stem cells and are a promising alternative to BMPs (1).  In this study, we study novel Oxy133's ability to induce osteogenesis and regenerate bone.

Methods: Rabbit BMSCs were isolated from femurs, cultured, and treated with control media, osteogenic media, various concentrations of Oxy133 (0, 0.1, 1, 5 μM), optimal concentration of Oxy49 (1uM), or optimal dose BMP-2. BMSCs were also conditioned with cyclopamine, a Hh signaling pathway inhibitor, followed by treatment with Oxy133. Osteogenic differentiation was assessed with alkaline phosphatase (ALP) assay and Alizarin Red staining. Critical-sized rabbit calvarial defects (6mm) were treated with either 1) collagen sponge with 10 mg Oxy133, 2) collagen sponge with 1 mg Oxy133, 3) collagen sponge incorporating inert vehicle (control), or 4) no treatment. The calvarium was harvested after seven weeks for histologic and radiographic analysis with micro-computed tomography.

Results:  ALP activity was significantly increased in rabbit BMSCs exposed to 0.1 and 1 µM of Oxy133 and to BMP-2. There was no significant difference between the ALP activities of Oxy49 (1 µM) and BMP-2 treatments. Alizarin red staining showed significantly greater calcium deposition in cells treated with Oxy133 or BMP-2 compared to controls. ALP activity produced by Oxy133 treatment was significantly inhibited by cyclopamine. Critical-sized rabbit calvarial defects showed significantly increased bone regeneration when treated with collagen sponges combined with Oxy133.

Conclusion:  Oxy133 is a potent osteo-inductive agent with higher efficacy than that of optimal dose BMP-2. It exerts its influence through activation of Hh signaling at an optimal concentration of 1uM, and is successful in healing critical sized rabbit calvarial defects. Oxy133 may be a more cost effective alternative to BMP-2 in the realm of bone regeneration.