A Metabolic Strategy Utilizing a Zein Scaffold for Bone Repair

项目摘要 在美国，每年进行超过一百万例手术，用于重建手术，创伤， 或异常的骨骼缺陷。为了达到重建目标，需要大量自体骨移植或 在外科手术中需要替代的大块同种异体移植物。自体骨供应有限， 同种异体骨材料和替代物例如合成移植物经常导致延迟的骨整合。 同种异体骨和合成替代物可以提供骨传导环境，但它们不提供 发生骨愈合和愈合所必需的细胞和/或其它生物活性。一种新的方法来 克服这些局限性的方法是使用代谢策略来促进愈合。我们小组最近的研究 已经确定氨基酸谷氨酰胺是成骨细胞特化和分化的关键调节剂， 间充质干细胞（MSC）。骨髓间充质干细胞是骨愈合和再生的关键，提供了一个水库 成骨细胞对损伤的反应。拟议的研究将利用富含谷氨酰胺的支架， 刺激内源性MSCs增殖分化为成骨细胞，并具有骨传导性， 骨缺损的骨修复。玉米醇溶蛋白是一种来源于玉米的蛋白质，其谷氨酰胺含量高，因此提供了一种抗氧化剂。 酶促降解后，谷氨酰胺的来源。它可以容易地处理，是生物相容的， 最近的研究表明，玉米醇溶蛋白通过相互作用增强了细胞附着 组织转氨酶我们首次证明了，我们可以形成水解稳定的， 纤维玉米蛋白支架。纤维是细胞粘附和生长的有益结构特征，这是由于大的 表面积与体积比和高纵横比，并且纤维结构模仿天然胶原蛋白的胶原纤维结构。 细胞外基质（ECM）。玉米醇溶蛋白也被美国食品和药物管理局归类为一般的 被公认为安全物质（GRAS），并正在寻求用于各种生物医学应用。这项建议 将开发用于骨再生的纤维玉米蛋白支架，目的是提供谷氨酰胺来源 来促进修复将处理以下具体目标。目的1将确定玉米醇溶蛋白支架， 促进MSC成骨。这项研究将检查玉米醇溶蛋白支架，随着交联程度的变化， 可以影响谷氨酰胺的利用率及其对MSC生长和成骨的影响。我们还将检查谷氨酰胺 通过谷氨酰胺酶（GLS）活性进行摄取和代谢，GLS是负责谷氨酰胺的主要酶 猫我们将抑制GLS活性以确定玉米醇溶蛋白的谷氨酰胺含量对分化的贡献。 目的2将研究玉米醇溶蛋白支架在骨缺损模型中的有效性。将在以下时间内评价骨愈合 时间我们还将使用LeprCre特异性敲除MSC中的GLS，并确定玉米醇溶蛋白支架的作用 关于骨骼愈合这项研究提出了一种新的纤维玉米醇溶蛋白支架修复骨缺损， 支持未来的研究，以验证导致临床转化的更大动物模型的结果。

Project Summary More than one million operations are performed annually in the United States for reconstructive surgery, trauma, or abnormal skeletal defects. To achieve reconstructive goals, large amounts of autologous bone graft or alternative large bulk allograft are needed in the surgical procedure. Autologous bone is limited in supply and allograft bone material and alternatives such as synthetic grafts often result in delayed osseous integration. Allograft bone and synthetic substitutes may provide an osteoconductive environment but they do not provide the necessary cellular and/or other biological activity for bony union and healing to occur. A novel approach to overcome these limitations is the use of a metabolic strategy to promote healing. Recent studies by our group have identified the amino acid glutamine as a critical regulator of osteoblast specification and differentiation in mesenchymal stem cells (MSCs). MSCs are critical for bone healing and regeneration by providing a reservoir of osteoblasts in response to injury. The proposed studies will utilize a glutamine enriched scaffold that can stimulate endogenous MSCs to proliferate and differentiate into osteoblasts and be osteoconductive to promote bone repair of skeletal defects. Zein, which is a protein derived from corn, is high in glutamine thus providing a source of glutamine to cells upon enzymatic degradation. It can be readily processed, is biocompatible and biodegradable and recent studies have demonstrated that cell attachment is enhanced on zein via interaction with tissue transglutaminase. We have demonstrated, for the first time, that we can form hydrolytically stable, fibrous zein scaffolds. Fibers are a beneficial structural feature for cell adhesion and growth due to the large surface-to-volume and high aspect ratio and the fibrous structure mimics the collagen fiber structure of the native extracellular matrix (ECM). Zein is also classified by the U.S. Food and Drug Administration as a generally recognized as safe substance (GRAS) and is being sought for a variety of biomedical applications. This proposal will develop fibrous zein scaffolds for use in bone regeneration with the goal of providing a source of glutamine to cells to promote repair. The following specific aims will be addressed. Aim 1 will determine zein scaffolds that promote MSC osteogenesis. The studies will examine zein scaffolds that vary with degree of crosslinking, which can affect glutamine availability, and its effect on MSC growth and osteogenesis. We also will examine glutamine uptake and metabolism via glutaminase (GLS) activity, which is the primary enzyme responsible for glutamine catabolism. We will inhibit GLS activity to determine the contribution of zein’s glutamine content on differentiation. Aim 2 will investigate the efficacy of zein scaffolds in a bone defect model. Bone healing will be evaluated over time. We will also knockout GLS specifically in MSCs using LeprCre and determine the effects of the zein scaffold on bone healing. This study proposes a novel fibrous zein scaffold for the repair of bone defects and findings will support future studies to validate results in larger animal models leading to clinical translation.