Bioengineering Single Crystal Growth

非技术摘要：经过数亿年的进化，生物矿化组织经常显示出非凡的性能水平。例如，骨骼在低重量下表现出高韧性，并且能够自我修复；有些无脊椎动物的牙齿即使连续磨损也能自磨。生物通常通过环境可持续的过程在温和的条件下形成这些材料，使它们成为生物启发或生物激活合成的有吸引力的目标。尽管自然界中有丰富的这类物质，但许多允许生物体控制它们形成的生物机制仍然知之甚少。作为一种模式生物，海胆对矿化具有显著的控制作用，形成光滑弯曲和分枝的碳酸钙（CaCO3）单晶内骨骼针状体。此前，该团队设计了一个海胆胚胎原代间充质细胞（PMCs）的体外培养系统，以在实验室中控制这些针状体的生长。他们发现一种信号蛋白VEGF控制着pmc沉积的针状体的形状。接下来，该团队将探索一些蛋白质的作用，这些蛋白质在转录组学实验中已被确定为潜在的晶体生长调节剂。技术包括蛋白质组学、微流控液滴反应器的体外分析和体内敲除实验。综上所述，这些研究将有助于提高对生物控制矿化的理解。在分子生物学、材料科学和生物工程的交叉点上，这项研究有可能为丰富的新技术提供信息，从生物启发和生物激活材料到二氧化碳封存材料。该团队将利用这项研究的跨学科潜力，培养来自广泛背景的本科生和研究生。本科研究助理将学习基本的生物实验室技能，同时也使用先进的材料表征技术。最后，该团队将利用作为美国国家科学基金会主要研究仪器拨款的一部分开发的仪器来帮助本科生可视化海水中的相变，从而扩大项目。我们的目标是基础材料科学教育。技术摘要：拟议的活动解决了生物体如何控制晶体生长过程的理解空白，长期目标是开发生物启发和生物激活材料。在之前的工作中，研究小组发现了VEGF信号在海胆胚胎初代间充质细胞（PMCs）沉积的方解石内骨骼单晶分支中的作用。展望未来，该团队将使用定量蛋白质组学来确认转录组学鉴定的蛋白质是否参与晶体生长控制。选择的蛋白质将重组生产，重组抗体（rAbs）将针对他们提出。rAbs将用于绘制穿过针状沉积囊泡和针状本身的蛋白质。与此同时，该团队将使用现有的微流控设备来分析天然蛋白和重组蛋白对成核、多晶选择和晶体生长的影响。最后，研究小组将用体内功能分析来补充这些实验。综上所述，该团队希望对相关蛋白质的表达、针状基质中的定位以及对成核动力学和晶体生长的影响之间的联系有一个详细的机制理解。这是将关键分子转化为更容易设计和扩展的系统的重要的第一步，使用合成生物学工具进行材料处理。因此，该团队将解决2012年美国国家科学基金会生物材料研讨会报告中确定的硬材料的所有四个挑战。作为研究目标的补充，该团队将让本科生参与研究，将研究成果纳入本科生实验室模块，并通过西北大学的社会科学项目招收高中实习生。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-technical abstract: Optimized during hundreds of millions of years of evolution, biomineralized tissues frequently display an extraordinary level of performance. Bone, for example, displays high toughness at low weight and is capable of self-repair; some invertebrate teeth self-sharpen despite continuous wear. Organisms typically form these materials under mild conditions through environmentally sustainable processes, making them appealing targets for bio-inspired or bio-enabled syntheses. Despite the abundance of such materials in nature, many biological mechanisms that allow the organism to control their formation remain poorly understood. As a model organism, sea urchins exert remarkable control over mineralization, creating smoothly curving and branched, yet single crystalline endoskeletal spicules of calcium carbonate (CaCO3). Previously, the team designed an in vitro culture system of sea urchin embryo primary mesenchyme cells (PMCs) to control the growth of these spicules in the laboratory. They discovered that a signaling protein, VEGF, controls the shape of spicules deposited by PMCs. The team will next probe the roles of a number of proteins that have been identified as potential crystal growth regulators in transcriptomics experiments. Techniques include, proteomics, in vitro assays in microfluidic droplet reactors, and knock-down experiments in vivo. In combination, these studies will lead to an improved understanding of biologically controlled mineralization. Poised at the intersection of molecular biology, materials science, and bioengineering, this research has the potential to inform a wealth of new technologies, from bio-inspired and bio-enabled materials to materials for carbon dioxide sequestration. The team will to leverage the interdisciplinary potential of this research to train undergraduate and graduate students from a broad range of backgrounds. An undergraduate research assistant will learn fundamental biological laboratory skills while also using advanced materials characterization techniques. Finally, the team will utilize instrumentation developed as part of a NSF Major Research Instrumentation grant to help undergraduate students visualize phase transformations in seawater, thus broadening the project?s reach to fundamental materials science education.Technical abstract: The proposed activities address gaps in the understanding of how living organisms control crystal growth processes, with the long-term objective to develop bio-inspired and bio-enabled materials. In prior work, the team discovered the role of VEGF signaling in branching of endoskeletal single crystals of calcite that are deposited by primary mesenchyme cells (PMCs) of the sea urchin embryo. Going forward, the team will use quantitative proteomics to confirm proteins that have been identified by transcriptomics as candidates involved in crystal growth control. Selected proteins will be produced recombinantly, and recombinant antibodies (rAbs) will be raised against them. rAbs will be used to map proteins across the spicule deposition vesicle and the spicule itself. In parallel, the team will use existing microfluidic devices to dissect the impact of native and recombinant proteins, individually and collectively on nucleation, polymorph selection, and crystal growth. Finally, the team will complement these experiments with functional analyses in vivo. Taken together, the team expects to develop a detailed mechanistic understanding of how expression of the relevant proteins, localization in the spicule matrix, and impact on nucleation kinetics and crystal growth may be connected. This is an important first step towards translating key molecular players into a system that is more easily engineered and scaled up, using the tools of synthetic biology for materials processing. The team will thereby address all four challenges in hard materials identified in the Report on the 2012 NSF Biomaterials Workshop. Complementary to the proposed research objectives, the team will engage undergraduates in research, incorporate research outcomes into undergraduate laboratory modules, and host high school interns through Northwestern University's Science in Society program.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.