Development of Osteogenic Oxysterols for Local Bone Formation

描述（由申请人提供）：我们之前已经报道了体内形成的作为胆固醇代谢中间体的特异性内源性产生的小分子氧固醇在体外对多能间充质干细胞（MSC）具有强效成骨作用。我们已经表明，这些影响是通过激活刺猬（Hh）信号通路介导的，独立于Hh蛋白。此外，我们已经证明，这些氧固醇诱导新生小鼠颅骨器官培养物中的骨形成，以及大鼠颅骨临界尺寸缺损模型和大鼠脊柱融合模型中的骨愈合。因此，这些氧固醇可以潜在地替代目前使用的昂贵的重组骨形态发生蛋白（BMP）和自体骨移植物，用于刺激非愈合性骨折和严重骨缺损中的局部骨形成。此外，我们已经表明，当与BMP组合使用时，成骨氧固醇大大降低了体外诱导有效成骨所需的BMP浓度。因此，我们最近进行了氧固醇结构活性关系（SAR）研究，导致合成了一个新的家族的有效的氧固醇类似物，具有成骨和抗脂肪形成活性等于或上级的母体氧固醇。然而，除了激活Hh途径之外，成骨氧固醇还激活肝脏X受体（LXR）信号传导，其在骨生成中的作用尚不清楚，并且可以通过调节Notch信号传导的靶点来促进氧固醇诱导的骨生成。因此，我们假设成骨氧化固醇类似物激活骨祖细胞中的Hh和LXR信号传导，并且这两种信号传导途径在对氧化固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）阐明成骨氧固醇类似物的分子信号传导谱，以确定Hh和LXR信号传导在体外氧固醇诱导的成骨中的作用和相对激活水平; 2）使用大鼠脊柱融合和股骨缺损模型检查氧固醇的成骨活性及其在局部给药的体内作用机制，同时定义了用于局部递送的最佳支架并阐明了Hh信号传导在体内氧化固醇效应中的作用;和3）通过确定Hh信号通过Gli转录因子是否介导氧固醇和BMP之间在体外和体内刺激骨生成中的协同作用，检查基线和氧固醇诱导的Hh途径活性在BMP 2诱导的骨生成中的作用，以及氧固醇是否可以通过抑制BMP 2对脂肪生成的刺激而潜在地改善新形成的骨的质量。这些研究将提供重要的新信息，有关的基本机制，调节成骨和氧化固醇的影响，以及指导未来的调查氧化固醇作为一个潜在的新类别的骨诱导剂局部和全身性骨再生。

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.