A Metabolic Strategy Utilizing a Zein Scaffold for Bone Repair

项目摘要 在美国，每年有100多万例重建手术、创伤手术、 或者不正常的骨骼缺陷。为了达到重建的目标，大量的自体骨移植或 在外科手术中需要替代的大体积异体移植。自体骨供应有限， 同种异体骨材料和替代物，如合成移植物，往往会导致延迟的骨整合。 同种异体骨和合成替代物可以提供骨传导环境，但它们不能提供 骨性愈合和愈合所必需的细胞和/或其他生物学活动。一种新的方法来 克服这些限制是使用新陈代谢策略来促进愈合。我们小组最近的研究 已确定氨基酸谷氨酰胺是成骨细胞规格和分化的关键调节因子 间充质干细胞。骨髓间充质干细胞是骨愈合和再生的关键，因为它提供了一个储存库。 成骨细胞对损伤的反应。拟议的研究将使用一种富含谷氨酰胺的支架，它可以 刺激内源性MSCs增殖分化为成骨细胞，并具有成骨促进作用 骨缺损的骨修复。玉米醇溶蛋白是一种从玉米中提取的蛋白质，它富含谷氨酰胺，因此提供了 谷氨酰胺是细胞在酶作用下降解的来源。它可以很容易地加工，具有生物兼容性和 可生物降解和最近的研究表明，细胞在玉米醇溶蛋白上的附着是通过相互作用而增强的 组织转谷氨酰胺酶。我们第一次证明了，我们可以形成稳定的， 纤维玉米醇溶蛋白支架。纤维是细胞黏附和生长的有益结构特征，因为 表面与体积之比和高纵横比以及纤维结构模仿了天然的胶原纤维结构 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 被公认为安全物质(GRAS)，正被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醇溶蛋白支架，目标是提供谷氨酰胺来源 以促进细胞修复。将实现以下具体目标。目标1将确定玉米醇溶蛋白支架 促进MSC成骨。这些研究将检查不同交联度的玉米醇溶蛋白支架， 可以影响谷氨酰胺的利用率，以及它对MSC生长和成骨的影响。我们还将检测谷氨酰胺 通过谷氨酰胺酶(GLS)的摄取和代谢，谷氨酰胺酶是谷氨酰胺的主要酶 分解代谢。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研究玉米醇溶蛋白支架在骨缺损模型中的作用。将对骨愈合情况进行评估 时间到了。我们还将使用LeprCre敲除MSCs中的GLS，并确定Zein支架的效果 关于骨愈合的研究。这项研究提出了一种用于修复骨缺损的新型纤维玉米醇溶蛋白支架，研究结果将 支持未来的研究，以验证在更大的动物模型中导致临床翻译的结果。

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.