Biomaterial Applications of Recombinant Bacterial Collagens

重组细菌胶原蛋白的生物材料应用

• 批准号: 8323975
• 负责人: BARBARA M BRODSKY
• 金额: $ 32.42万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8323975
• 关键词: Active Sites; Affinity; Animals; Bacteria; Bacterial Proteins; Basic Science; Beta-N-Acetylglucosaminidase; Binding; Biocompatible Materials; Biological; Bone Marrow; Cell Line; Cells; Cellular biology; Coiled-Coil Domain; Collagen; DDR2 gene; Data; Dendritic Cells; Development; Differentiation and Growth; Enzymes; Escherichia coli; Evaluation; Fibrillar Collagen; Film; Fostering; Generations; Growth; Higher Order Chromatin Structure; Human; Hydroxyproline; Implant; In Vitro; Inflammatory Response; Integrins; Length; Matrix Metalloproteinases; Mechanics; Mediating; Mesenchymal Stem Cells; Methodology; Modification; Molecular; Morphology; Mus; Osteoblasts; Porifera; Property; Proteins; Receptor Cell; Recombinants; Signal Transduction; Site; Specificity; Stem cells; Streptococcus pyogenes; Structure; System; Tissue Engineering; Variant; Work; animal tissue; base; bone; crosslink; design; experience; genetic manipulation; human stem cells; in vitro testing; in vivo; large scale production; macrophage; novel strategies; osteogenic; product development; protein degradation; public health relevance; receptor; response; scaffold; stem cell differentiation; subcutaneous; tool; triple helix

DESCRIPTION (provided by applicant): There is a need for well-defined standardized collagen proteins amenable to easy sequence modifications and large scale production levels for biomedical and biomaterial applications. This proposal aims to express recombinant bacterial collagens in high yield in E. coli to use both as a basic science tool for characterizing biologically active collagen domains and as a scaffold to promote bone generation by human stem cells. Bacterial collagen proteins expressed from S. pyogenes and four other bacteria in high yield have been shown to form triple-helical molecules of high stability despite the absence of hydroxyproline. Higher order structural aggregates formed by bacterial collagens will be characterized and cross-linked for stability, and the mechanical properties of materials formed will be characterized. Collagen variants will also be designed to contain a trimmer coiled coil adjacent to the bacterial collagen-like domain to expand the opportunities for increased molecular stability, formation of heterotrimeric molecules to foster protein association, and the inclusion of specific cell binding and matrix metalloproteinase (MMP) cleavage sites in order to better define collagen cell receptors and control turnover of the protein. The ability of tailored bacterial collagen proteins to support growth and differentiation of human bone marrow derived mesenchymal stem cells as potential biomaterials for bone replacement will be investigated. Bacterial collagens with and without inserted biological signals will be compared with extracted mammalian collagens using a range of osteogenic phenotypic and genotypic markers. In addition, inflammatory responses to the various collagens will be compared in vitro and in vivo to animal collagens using macrophage and dendritic cell screens, and subcutaneous implants in mice. These comparisons will provide baseline data on biological responses at the cell, tissue and animal level to the collagen variants. High yields and straightforward genetic manipulations in this system will allow combinations of different biologically active sites and permit interactive optimization to design biomaterials of appropriate structural and biological properties to fine tune stem cell responses related to basic cell biology and applied biomaterials and tissue engineering needs. The interdisciplinary team of collaborators brings important complementary expertise to make this project successful, with Dr. Brodsky's extensive work on collagen structure, Dr. Kaplan's track record with stem cells and biomaterials, Dr. Ramshaw's experience with collagen, biomaterials and product development, and our consultants expertise in matrix metalloproteinases (Dr. Nagase) and DDR receptors (Dr. Leitinger). PUBLIC HEALTH RELEVANCE: There is a need for well-defined standardized collagen proteins amenable to easy sequence modifications and large scale production for biomedical and biomaterial applications. The development of a bacterial collagen system to incorporate biologically active sites and to form well-defined hierarchical structures would revolutionize the use of this important protein in both fundamental and applied studies.

描述(由申请人提供)：生物医学和生物材料应用需要定义明确的标准化胶原蛋白，易于进行序列修改和大规模生产。本研究的目的是在大肠杆菌中高效表达重组细菌胶原蛋白，作为鉴定具有生物活性的胶原蛋白结构域的基础科学工具，以及作为促进人类干细胞成骨的支架。在化脓性链球菌和其他四种细菌中高效表达的细菌胶原蛋白已被证明在不含羟基脯氨酸的情况下仍能形成高度稳定的三螺旋分子。将对细菌胶原蛋白形成的高阶结构聚集体进行表征，并对其稳定性进行交联，并对形成的材料的力学性能进行表征。胶原蛋白变体还将被设计为在细菌胶原样结构域附近包含一个微调螺旋，以扩大提高分子稳定性的机会，形成异三聚体分子以促进蛋白质结合，并包含特定的细胞结合和基质金属蛋白酶(MMP)裂解部位，以便更好地定义胶原细胞受体和控制蛋白质的周转。定制的细菌胶原蛋白支持人骨髓间充质干细胞作为潜在的骨替代生物材料的生长和分化的能力将被研究。有和没有插入生物信号的细菌胶原蛋白将使用一系列成骨表型和基因标记与提取的哺乳动物胶原蛋白进行比较。此外，使用巨噬细胞和树突状细胞筛选，以及小鼠皮下植入，将在体外和体内将各种胶原蛋白的炎症反应与动物胶原蛋白进行比较。这些比较将提供在细胞、组织和动物水平上对胶原蛋白变体的生物反应的基线数据。该系统的高产量和简单的遗传操作将允许不同生物活性部位的组合，并允许交互优化设计具有适当结构和生物特性的生物材料，以微调与基础细胞生物学、应用生物材料和组织工程需求相关的干细胞反应。合作伙伴的跨学科团队为该项目的成功提供了重要的互补专业知识，Brodsky博士在胶原结构方面的广泛工作，Kaplan博士在干细胞和生物材料方面的记录，Ramshaw博士在胶原、生物材料和产品开发方面的经验，以及我们的顾问在基质金属蛋白酶(Nagase博士)和DDR受体(Leitinger博士)方面的专业知识。 公共卫生相关性：需要定义明确的标准化胶原蛋白，易于序列修改和大规模生产，用于生物医学和生物材料应用。细菌胶原蛋白系统的发展将结合生物活性部位并形成明确的分级结构，这将彻底改变这种重要蛋白质在基础和应用研究中的使用。