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“金标准”是自体骨移植物，但自体移植物受到可用性和供体部位发病率的限制。各种基于骨诱导生长因子的疗法已经被开发出来，试图找到一种有效和安全的骨再生方法。在各种骨诱导因子中，骨形态发生蛋白（BMP）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，并且其超生理剂量需求导致不利的副作用，例如囊肿形成和低效的骨形成。因此，需要开发替代的骨诱导生长因子策略，可以有效补充BMP活性，以最大限度地提高生物学效率，同时最大限度地减少BMP剂量。 一种替代方法是根本不递送BMP，同时增强参与再生的祖细胞响应内源性BMP的能力。这可以通过递送BMP拮抗剂的抑制剂如Noggin来实现，从而增强内源性BMP活性。头蛋白是骨祖细胞中BMP途径的直接靶标，因此很可能临床上需要超生理学BMP剂量，这在很大程度上是由于头蛋白诱导。因此，我们提出了一种通过下调Noggin来增强BMP信号传导的方法。内源性BMP的效力可以通过递送比BMP更具特异性和更少多效性的骨诱导信号来增强，例如Nell-1 [Nel样分子-1; Nel（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）]。在以前的研究中，Nell-1已被证明可以加速体外成骨分化和体内颅骨骨形成。此外，Nell-1是一种分泌蛋白，可以在细胞外递送，最重要的是，Nell-1促进与BMP 2对骨再生的协同作用。通过局部抑制Noggin，我们寻求增强内源性BMP信号传导，其反过来应协同刺激由Nell-1诱导的成骨细胞分化，从而导致最大骨形成，而无需担心BMP介导的副作用。该提议的具体假设是，Nell-1的控制递送结合Noggin抑制剂的使用可以增强节段性股骨缺损的修复。两个具体的目标，提出了调查这一假设。目的1：通过Noggin抑制+ Nell-1增强骨再生。在这个特定的目标中，我们将评估Nell-1与Noggin抑制的MSC组合对成骨能力和骨再生的协同作用。目的2：通过Noggin-siRNA + Nell-1的受控递送来增强骨再生。在这一目标中，我们将开发释放Nell-1和Noggin-siRNA的非病毒基因递送/支架系统，并将测试它们是否可以在节段性股骨缺损模型中有效地再生骨。

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.