Research Project
10109396
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.
