Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi-synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone-healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP-2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP-2 were used to reconstruct critical-sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μm Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μm Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd.

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