Development of Osteogenic Oxysterols for Local Bone Formation

描述（由申请人提供）：我们之前报道过特异性内源性小分子氧甾醇，作为体内胆固醇代谢的中介，在体外对多能间充质干细胞（MSC）具有强大的成骨作用。我们已经证明，这些作用是通过激活独立于Hh蛋白的Hedgehog （Hh）信号通路介导的。此外，我们已经证明这些氧化甾醇在新生小鼠颅骨器官培养中诱导骨形成，并在大鼠颅骨临界尺寸缺陷模型和大鼠脊柱融合模型中诱导骨愈合。因此，这些氧甾醇有可能取代目前使用的昂贵的重组骨形态发生蛋白（BMPs）和自体骨移植物来刺激未愈合骨折和严重骨缺损的局部骨形成。此外，我们已经证明，当与bmp联合使用时，成骨氧甾醇大大降低了体外诱导有效成骨所需的bmp浓度。因此，我们最近进行了氧甾醇结构活性关系（SAR）研究，合成了一种新的强效氧甾醇类似物家族，具有与母体氧甾醇相同或优于母体氧甾醇的成骨和抗脂肪活性。然而，除了激活Hh通路外，成骨氧甾醇还激活肝脏X受体（LXR）信号，其在成骨中的作用尚不清楚，可能通过调节Notch信号的靶点来促进氧甾醇诱导的成骨。因此，我们假设成骨氧甾醇类似物激活骨祖细胞中的Hh和LXR信号通路，并且这两种信号通路在对氧甾醇的成骨反应中起重要作用。为了验证这一假设，我们将追求以下具体目标：1)阐明成骨性氧甾醇类似物的分子信号谱，以确定Hh和LXR信号在体外氧甾醇诱导成骨中的作用和相对激活水平；2)利用大鼠脊柱融合术和股骨缺损模型，研究氧甾醇的成骨活性及其在体内局部给药的作用机制，同时确定局部给药的最佳支架，并阐明Hh信号在体内氧甾醇作用中的作用；3)通过确定通过Gli转录因子的Hh信号传导是否介导氧甾醇和bmp在体外和体内刺激成骨过程中的协同作用，以及氧甾醇是否可能通过抑制BMP2刺激脂肪生成来改善新生骨的质量，检验基线和氧甾醇诱导的Hh通路活性在bmp诱导成骨过程中的作用。这些研究将提供关于调控成骨的基本机制和氧甾醇作用的重要新信息，并为今后研究氧甾醇作为一种潜在的新类型的局部或全身性成骨诱导剂提供指导。

DESCRIPTION (provided by applicant): We have previously reported that specific endogenously produced small molecule oxysterols, formed in vivo as intermediaries of cholesterol metabolism, have potent osteogenic effects on multipotent mesenchymal stem cells (MSC) in vitro. We have shown that these effects are mediated through activation of the Hedgehog (Hh) signaling pathway, independent of Hh proteins. In addition, we have demonstrated that these oxysterols induce bone formation in neonatal mouse calvaria organ cultures, and bone healing in both a rat calvarial critical-sized defect model and a rat spinal fusion model in vivo. Thus, these oxysterols could potentially replace currently used expensive recombinant bone morphogenetic proteins (BMPs) and autogenous bone grafts for stimulation of local bone formation in non-healing fractures and critical bone defects. In addition, we have shown that when used in combination with BMPs, osteogenic oxysterols greatly reduce the concentration of BMPs required to induce effective osteogenesis in vitro. Accordingly, we have recently performed oxysterol structure activity relationship (SAR) studies that have resulted in the synthesis of a novel family of potent oxysterol analogues that possess osteogenic and anti-adipogenic activity equal or superior to the parent oxysterols. However, in addition to activating the Hh pathway, osteogenic oxysterols also activate liver X receptor (LXR) signaling whose role in osteogenesis is unclear and may promote oxysterol-induced osteogenesis by regulating targets of Notch signaling. Therefore, we hypothesize that osteogenic oxysterol analogues activate Hh and LXR signaling in osteoprogenitor cells and that both signaling pathways play an important role in osteogenic responses to oxysterols. To examine this hypothesis, we will pursue the following specific aims: 1) Elucidate the molecular signaling profile of osteogenic oxysterol analogues to determine the role and relative activation levels of Hh and LXR signaling in oxysterol-induced osteogenesis in vitro; 2) Examine the osteogenic activity of oxysterols and their mechanism of action in vivo for local administration using the rat spinal fusion and femoral defect models, while defining the optimal scaffold for local delivery and elucidating the role of Hh signaling in oxysterol effects in vivo; and 3) Examine the role of baseline and oxysterol-induced Hh pathway activity in BMP2-induced osteogenesis by determining whether Hh signaling through Gli transcription factors mediates the synergy between oxysterols and BMPs in stimulating osteogenesis in vitro and in vivo, and whether oxysterols may potentially improve the quality of the newly formed bone by inhibiting BMP2 stimulation of adipogenesis. These studies will provide important new information regarding the basic mechanisms regulating osteogenesis and the effects of oxysterols, as well as guidance for future investigations of oxysterols as a potential new class of osteoinductive agents for localized and perhaps systemic osteoregeneration.