INTELLECTUAL MERIT: Despite the highly attractive properties of biological mineralized tissues and great recent progress in bio-inspired materials synthesis, many of the hallmarks of biological crystal growth have yet to be reproduced in vitro: polymorph control, curving and/or branching single crystals, and nm scale control of organic-inorganic composites. Much could be gained by developing a biotechnological alternative to bulk materials synthesis. In this proposal the PI will explore micro-patterned cell culture of sea urchin embryonic primary mesenchyme cells (PMCs) as a means to guide the cellular machinery responsible for single crystal synthesis. In this way the project will take advantage of a fully capable synthetic engine and attempt to reprogram it. This will then provide the basis for investigating and ultimately reverse engineering it in vitro. The following specific tasks will be pursued to explore and develop the range and limits of engineering single crystal shape, connectivity, and larger composite structures by guided biological deposition: (1) Develop micro-patterning to control the placement of PMCs on a surface. In this way, guide the biological deposition of single calcite (CaCO3) crystals. In particular, establish pattern topography, adhesiveness, chemotaxis induced by vascular endothelial growth factor (VEGF), and limiting cell numbers and densities. (2) Explore junction patterns to reproduce the biological tri-radiate branching or fusion of two individual single crystalline elements. Combine simple shapes and junctions for increasingly complex crystal shape and connectivity in 2D and 3D. (3) Further characterize the biological crystal growth machinery with respect to routes of mineral/protein trafficking, amorphous precursor phases, and amorphous-to crystalline transitions by a complementary suite of techniques including live cell imaging, IR and Raman microscopy, and electron microscopic imaging.
BROADER IMPACTS: The proposed research promises a major advance in harnessing biosynthetic mineralization pathways to create useful ceramic structures. The project also includes a thorough plan for integration of research and education. The PI will develop a full quarter undergraduate course in biomineralization designed to train students in communications skills, expose them to professional peer review, and assess their analytical skills. Students will disseminate the knowledge gained through an online, public access WIKI that will be written, reviewed, and edited by students. A design competition will be held to develop new cell culture devices for guided crystal growth and thus draw students into questions actively researched in the PI?s laboratory and engage their engineering skills in a trans-disciplinary project. Undergraduate research opportunities in the PI?s lab will provide highly interdisciplinary cross training in bio-related areas such as sea urchin husbandry, cell culture, and live cell imaging, and these activities will complement training in clean room photolithography and materials characterization by advanced imaging techniques. Outreach to high school students will be targeted to ethnically diverse neighborhoods of Chicago, Rogers Park, and Evanston. As all current graduate students in the lab are female, an especially focused projection of positive female role models will be achieved. In conjunction with high school science teachers, engineering and biomaterials class modules, visits on the Northwestern Campus, and a summer internship program will be organized.
Biomineralization is nature’s bottom-up nanofabrication process that yields nanoscale organic-inorganic composites with unrivaled structural control at all hierarchy levels. As a result, biominerals often possess mechanical properties superior to their synthetic counterparts. Harnessing this biosynthesis mechanism will provide a biotechnological alternative to materials synthesis as well as inspiration for the development of advanced materials. However, most biominerals are deposited only within living tissues and hence cannot be easily reproduced in vitro. A rare exception is the sea urchin embryo primary mesenchyme cells (PMC) which retain their ability to form linear spicules of single crystalline calcite (CaCO3) even when cultured in vitro. To control the formation and growth of single crystalline spicules in vitro, we have designed micro-patterned surfaces with patches of "sticky" proteins (lectins) that bind specifically to the PMC. We show that PMC bind to these patterns and deposit linear calcite spicules that are aligned with the underlying lectin template and oriented with the long-axis of spicules parallel to the calcite c-axis (Figure 1). In summary, this study not only provides an excellent tool for investigating the cellular deposition machinery in vitro, but also enables a bottom-up biotechnological approach for synthesizing oriented, single crystalline materials. Organisms such as the sea urchin embryo have a remarkable ability to control the structure and properties of biominerals. An organic matrix comprised of biomacromolecules frequently interacts with the forming mineral. For example, in the tooth of the chiton, a marine invertebrate, the organic matrix, composed of the sugars and proteins, controls the mineral formation. As the tooth matures, the organic matrix is incorporated into mineral. The outstanding toughness and wear resistance of the tooth results from the organic-inorganic interfaces that deflect and arrest cracks. An in-depth understanding of biological mineral formation and the advanced material properties resulting from interfaces depends on the ability to characterize the nanoscale interfaces. We pioneered the use atom probe tomography (APT) to characterize these interfaces. APT enables identification of individual atoms and molecules and pin-points their location within the sample in 3D, with a resolution better than 0.2 nm. We observed numerous nanoscale organic fibers within the chiton tooth and mapped the chemistry across the interface (Figure 2). This allowed us to discover a new chemical level of hierarchy in the chiton tooth. The implications of this discovery reach from the understanding of biominerals such as our bone and teeth to the discovery of new materials. Single crystals for technological applications are predominantly processed using "top down" fabrication methods, in which a large single crystal is first grown and then cut and etched to obtain the desired micro and nano-structures. In contrast, biology demonstrates sophisticated examples of "bottom up" synthesis, in which the size, shape, and orientation of a crystal are regulated as it grows. To assert this control, biology uses an unstable and disordered precursor that can be molded prior to crystallization. While crystal growth is traditionally performed in bulk, biological processing of these amorphous prescursors frequently occurs in specialized vesicles, tens of nanometers to micrometers in size. We work to understand how in biology vesicles form, stabilize, and shape the mineral precursors. Towards this goal, we developed a synthetic model. We first form vesicles containing calcium, and create calcium carbonate mineral by allowing carbon dioxide to diffuse inside. Using X-ray scattering and electron microscopy (Figure 3), we demonstrate the formation of stable nanoparticles of amorphous calcium carbonate (ACC). This unstable precursor is prevented from crystallizing due to confinement by the vesicle. Vesicle-stabilized ACC offers a unique platform to further study the effects of particle size, membrane chemistry, and additives in crystal synthesis. An understanding of how organisms use these parameters to control mineral growth could enable more efficient material syntheses with much greater control over material properties.
