Development of Osteogenic Oxysterols for Local Bone Formation

描述(申请人提供)：我们之前曾报道，体内形成的特定内源性小分子氧合固醇作为胆固醇代谢的中介，在体外对多潜能间充质干细胞(MSC)具有强大的成骨作用。我们已经证明，这些效应是通过激活Hedgehog(HH)信号通路而介导的，而不是HH蛋白。此外，我们还证明了这些氧化甾醇在新生小鼠颅骨器官培养中诱导骨形成，并在体内大鼠颅骨临界大小缺损模型和大鼠脊柱融合模型中诱导骨愈合。因此，这些氧化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)和自体骨移植来刺激不可愈合的骨折和严重的骨缺损的局部骨形成。此外，我们已经证明，当与BMPs联合使用时，成骨氧合固醇可极大地降低体外诱导有效成骨所需的BMPs浓度。因此，我们最近进行了氧固醇结构活性关系(SAR)研究，结果合成了一类新的强效氧固醇类似物，它们具有与母体氧固醇相同或更强的成骨和抗脂肪活性。然而，除了激活HH途径外，成骨氧固醇还激活肝X受体(LXR)信号，其在成骨中的作用尚不清楚，并可能通过调节Notch信号的靶点来促进氧固醇诱导的成骨。因此，我们假设成骨氧合固醇类似物激活了成骨前体细胞中的HH和LXR信号，并且这两条信号通路在成骨反应中起着重要的作用。为了验证这一假说，我们将追求以下特定目标：1)阐明成骨氧合固醇类似物的分子信号谱，以确定HH和LXR信号在氧合固醇诱导的体外成骨中的作用和相对激活水平；2)利用大鼠脊柱融合和股骨缺损模型，在体内检测局部给药的成骨活性及其作用机制，同时确定局部给药的最佳支架，并阐明HH信号在体内氧化甾醇效应中的作用；3)通过检测GLI转录因子介导的HH信号转导通路在体内和体外刺激成骨方面的协同作用，以及是否通过抑制BMP2对成骨的刺激而潜在地改善新骨的质量，来检测基础状态和氧合固醇诱导的HH途径在BMP2诱导的成骨中的作用。这些研究将为调控成骨的基本机制和氧合甾醇的作用提供重要的新信息，并为将来氧合甾醇作为一类潜在的局部或全身骨再生的骨诱导剂的研究提供指导。

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.