INTELLECTUAL MERIT: Biological mineralized tissues (e.g. bones, teeth, shells) are sophisticated organic-inorganic composites with hierarchical architecture down to the nm-scale. Their functional roles include mechanical strengthening, optical elements, and gravity or magnetic field sensing. Optimized in hundreds of millions of years of evolution, organisms have achieved remarkable engineering feats such as high bone toughness at low weight, self-sharpening teeth, self-repair capability, and low energy footprint, sustainable syntheses. Many of the hallmarks of biological crystal growth have yet to be reproduced in vitro: curving and/or branching single crystals, control over polymorph, use of amorphous precursors, and nm scale control of organic-inorganic composites. This project investigates approaches to control the cooperative growth of single crystals in cell culture with the goal to quite literally grow materials with properties optimized for their intended use. The biomineralization system in sea urchin serves as the platform for these studies. The following specific tasks will be pursued to explore and develop the range and limits of bioengineering single crystal shape, connectivity, and larger composite structures by guided biological deposition: (1) Explore, by multi-dimensional live cell microscopy, cellular dynamics on micro-patterns to better understand phenomena of bridging between sticky patches, motility of cells on lectin patterns, and formation of branch and/or fusion sites. (2) Characterize interplay of spicule mineral, organic matrix, and cellular machinery at key points in the spicule development, namely the initial deposit, linear spicules, branch sites, and joints. (3) Overexpress and purify the endogenous signaling factor SpVEGF-3 as a universal inducer of spiculogenesis in primary mesenchyme cell (PMC) cultures, and explore immobilization of the VEGF to study its role as a chemo-attractant. (4) Investigate spicule matrix protein sorting signals in PMCs and determine minimum signal peptide required to target proteins to the spicule compartment. (5) Use fusion proteins to study spicule matrix, and attempt to influence polymorph selection by expression of exogenous polymorph switches such as Starmaker.
BROADER IMPACTS: The ability to grow novel functional materials at ambient temperatures from seawater could create entirely new approaches to sustainable materials synthesis. In addition, the PI will expose freshmen and sophomores to sea urchin biomineralization and soft lithography in the context of new ?Discovery Lab? modules that are part of an entry-level materials science class. From this class and through minority recruitment channels at centers such as the MRSEC and others, he will recruit undergraduate researchers for academic year and summer research projects. Such students receive highly interdisciplinary training in bio-related areas (e.g. molecular biology, biochemistry, and cell culture) that will complement clean room training, photo and soft lithography, and materials characterization by cutting-edge techniques. The project will continue to develop modules for the mobile lab jump-started with the previous award to carry aspects of this research to local high and middle schools. In addition, a murder mystery activity that is being developed with the Chicago Botanic Garden will be adapted for use at the Shedd Aquarium. Finally, the PI will leverage resources at Northwestern University to reach out to society at large through participation in a range of events that include lab tours and public lectures.