Biomaterial Applications of Recombinant Bacterial Collagens

描述（由申请人提供）：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。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，作为表征生物活性胶原结构域的基础科学工具，并作为促进人类干细胞成骨的支架。由化脓性葡萄球菌和其他四种细菌高产表达的细菌胶原蛋白已被证明在缺乏羟基脯氨酸的情况下形成高稳定性的三螺旋分子。由细菌胶原形成的高阶结构聚集体将被表征并交联稳定性，形成的材料的机械性能将被表征。胶原蛋白变体也将被设计成包含一个修剪器，卷曲在细菌胶原样结构域附近，以扩大增加分子稳定性的机会，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）切割位点，以便更好地定义胶原细胞受体和控制蛋白质的周转。量身定制的细菌胶原蛋白支持人骨髓间充质干细胞生长和分化的能力，作为潜在的骨替代生物材料将被研究。利用一系列成骨表型和基因型标记，将细菌胶原与提取的哺乳动物胶原进行比较。此外，通过巨噬细胞和树突状细胞筛选，以及小鼠皮下植入，将各种胶原蛋白在体外和体内与动物胶原蛋白的炎症反应进行比较。这些比较将提供细胞、组织和动物水平上对胶原蛋白变异的生物学反应的基线数据。在该系统中，高产量和直接的遗传操作将允许不同生物活性位点的组合，并允许交互优化来设计适当结构和生物特性的生物材料，以微调与基本细胞生物学和应用生物材料和组织工程需求相关的干细胞反应。跨学科合作团队带来了重要的互补专业知识，使该项目取得成功，其中包括Brodsky博士在胶原蛋白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博士在胶原蛋白、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。

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.