Combined Effect of Noggin Suppression and Nell-1 on Bone Regeneration

描述(由申请人提供)：目前骨移植材料的“黄金标准”是自体骨移植，但自体骨移植受到可获得性和供区发病率的限制。各种基于骨诱导生长因子的治疗方法已经被开发出来，试图找到一种有效和安全的骨再生方法。在现有的各种骨诱导因子中，骨形态发生蛋白(BMPs)被认为是最有效的骨诱导因子，并已被广泛研究用于治疗许多骨折和骨缺损。然而，BMP是高度多效性的分子，其超生理剂量要求会导致不良副作用，如囊性形成和低效的骨形成。因此，有必要开发替代的骨诱导生长因子策略，能够有效地补充BMP的活性，以最大限度地提高生物学效率，同时将BMP剂量降至最低。一种替代方法是根本不传递BMP，同时增强参与再生的祖细胞对内源性BMP做出反应的能力。这可以通过输送BMP拮抗剂的抑制剂如Noggin来实现，从而增强内源性BMP的活性。Noggin是BMP途径在骨祖细胞中的直接靶点，因此临床上很可能需要超生理剂量的BMP，这在很大程度上是由于Noggin的诱导。因此，我们提出了一种通过下调Noggin来增强BMP信号的方法。内源性BMPs可以通过传递比BMPs更具特异性和更少多效性的骨诱导信号来增强其效力，例如Nell-1[NEL样分子-1；NEL(一种在神经组织中强烈表达的编码表皮生长因子样结构域的蛋白质)]。以前的研究表明，Nell-1在体外可以促进成骨分化，在体内可以促进颅骨的形成。此外，Nell-1是一种分泌蛋白，可以在细胞外传递，最重要的是，Nell-1促进了与BMP2在骨再生方面的协同效应。通过局部抑制Noggin，我们寻求增强内源性BMP信号，进而协同刺激Nell-1诱导的成骨细胞分化，从而导致最大限度的骨形成，而无需担心BMP介导的不良影响。这一建议的具体假设是，Nell-1的控制性释放结合Noggin抑制的使用可以促进股骨节段性缺损的修复。提出了两个具体的目标来研究这一假说。目的1：通过Noggin抑制+Nell-1促进骨再生。为此，我们将评估Nell-1联合Noggin抑制的MSC在成骨能力和骨再生方面的协同作用。目的2：通过控制输送Noggin-siRNA+Nell-1促进骨再生。为此，我们将开发释放Nell-1和Noggin-siRNA的非病毒基因传递/支架系统，并将测试它们是否能够有效地在股骨节段性缺损模型中再生骨。

DESCRIPTION (provided by applicant): The current "gold standard" for bone graft material is autologous bone graft, but autologous grafts are limited by availability and donor site morbidity. Various osteoinductive growth factor-based therapies have been developed in an attempt to find an effective and safer method of bone regeneration. Among the various osteoinductive factors available, bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors and have been extensively studied for the treatment of many bone fractures and bone defects. However, BMPs are highly pleiotropic molecules and their supra-physiological dose requirement leads to adverse side effects such as cyst formation, and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity to maximize biological efficiency while minimizing the BMP dose. One alternative approach is to deliver no BMP at all, while enhancing the ability of the progenitor cells that participate in regeneration to respond to endogenous BMPs. This can be accomplished by delivering inhibitors of BMP antagonists such as Noggin, thereby enhancing endogenous BMP activities. Noggin is a direct target of BMP pathways in osteoprogenitors, and it is thus highly likely that supraphysiological BMP doses are required clinically in large part due to Noggin induction. Thus, we propose an approach for enhancing BMP signaling through down-regulation of Noggin. The potency of endogenous BMPs can be enhanced by delivering osteoinductive signals that are more specific and less pleiotropic than BMPs, such as Nell-1 [Nel-like molecule-1; Nel (a protein strongly expressed in neural tissue encoding epidermal growth factor like domain)]. In previous studies, Nell-1 has been shown to accelerate osteogenic differentiation in vitro and calvarial bone formation in vivo. Moreover, Nell-1 is a secreted protein that can be delivered extracellularly, and most importantly Nell-1 promotes synergistic effects with BMP2 on bone regeneration. By suppressing Noggin locally, we seek to enhance endogenous BMP signaling which in turn, should synergistically stimulate osteoblast differentiation induced by Nell-1, thereby leading to maximum bone formation without the concerns surrounding BMP mediated adverse effects. The specific hypothesis of this proposal is that controlled delivery of Nell-1 combined with the employment of Noggin suppression can enhance repair of segmental femoral defects. Two specific aims are proposed to investigate this hypothesis. Aim 1: To enhance bone regeneration via Noggin suppression + Nell-1. In this specific aim, we will evaluate synergistic effects of Nell-1 combined with Noggin-suppressed MSC on osteogenic capacity and bone regeneration. Aim 2: To enhance bone regeneration via controlled delivery of Noggin-siRNA + Nell-1. In this aim, we will develop non-viral gene delivery/scaffolding systems that release Nell-1 and Noggin-siRNA and will test whether they can effectively regenerate bone in a segmental femoral defect model.