INTELLECTUAL MERIT: The sandcastle worm Phragmatopoma californica resides for physical protection in a composite mineral shell in the intertidal zone along the coast of California. The worm gathers the mineral component adventitiously as sand grains and bits of seashell hash which it bonds together into a tube with small dabs of a proteinaceous glue in the manner of building a stone wall. The glue sets within 30 s under cold seawater and hardens to a tough leathery consistency through oxidative crosslinking. Technological interest in this bioadhesive stems from its ability to bond a diverse range of mineral substrates under water. The adhesive is secreted as a colloidal suspension with low initial viscosity and interfacial tension that readily spreads on wet substrates, yet it is sufficiently cohesive that it does not disperse into the ocean before setting. Moreover, the water-borne bioadhesive apparently displaces interfacial water, a prerequisite for strong surface adhesion. These properties make the P. californica adhesive a valuable new paradigm for the design of water-borne, underwater adhesives. Several questions remain to be answered about the composition and biological processing of the adhesive. To illustrate, the adhesive gland contains at least two distinct types of secretory granules. (1) How are the glue proteins distributed between the granule types? (2) Are the glue proteins of each granule type expressed in distinct regions of the adhesive gland? (3) Does the mixing of the granule contents during secretion trigger the setting and curing reactions. Experiments have been devised to analyze the composition and biological processing of the adhesive before it sets. The first set of experiments (aim 1) will provide a broad overview of adhesive gland physiology through gene expression analysis. The second set of experiments (aim 2) will use the genetic and antibody probes resulting from aim 1 to investigate functional partitioning of the adhesive gland by examining spatial expression patterns. The third set of experiments (aim 3) will deploy powerful methods and modern mass spectrometry instrumentation to analyze the protein content of freshly secreted adhesive and of individual secretory granules. Peptides will be proteolytically generated and analyzed by micro-liquid chromatography and tandem mass spec (LC/MS/MS). Proteomic analysis will provide direct evidence that the new glue proteins discovered in the expression survey are present in the glue.
BROADER IMPACTS: The results of these studies will make progress towards solving important technological issues such as 'wet bonding' of bone-to-bone or bone-to-metal in implants using injectable and biocompatible adhesives as well as other 'under water' adhesive applications." Integration of teaching and research is achieved in a novel and effective way. The majority of the P. californica adhesive gland EST (expressed sequence tags) database will be developed during an undergraduate laboratory course in Molecular Bioengineering where students learn about construction cDNA libraries, purifying plasmids, automated DNA sequencing, bioinformatics resources, and how these tools are used to investigate biological processes. Students find this experience quite unique compared to canned laboratory exercises. The results are not known before hand, and the students have an authentic opportunity to discover new biotechnological resources. Students take ownership of the genes they are investigating, pursue the bioinformatics analysis, and develop hypotheses about the potential role of their gene in the bioadhesive.
Sandcastle worms are reef-building tubeworms. Their intertidal reef colonies can extend continuously for 100s of miles parallel to the shore, and occur along tropical and temperate coastlines worldwide. The wave-resistant structures have a major impact on stabilization and evolution of the beach environment by dissipating wave energy, filtering and trapping sediments, and providing habitats for a rich fauna. The massive reefs consist of side-by-side tubes about an 1/8 inch in diameter, each built by and home to an individual worm. The worms build their tubular homes by gluing together sandgrains and broken bits of seashells with an underwater adhesive, much like a mason would build a stone house. The turbulent shoreside building sites require robust construction, which attests to the durability of the water-proof adhesive bonds holding the composite tubes together. Intellectual merit. Development of effective manmade underwater adhesives remains elusive. Though hardware stores stock products described as "marine" adhesives, the directions invariable instruct the user to clean and dry the surface before application. Yet in nature, aquatic organisms like the sandcastle worm routinely produce robust adhesive bonds while completely submerged in water. The objective and intellectual merit of the NSF sponsored project was to decipher the biological processing pathways, the composition, and chemistry of the natural sandcastle worm adhesive. This knowledge and understanding has guided development of practical and cost effective synthetic adhesives for bonding wet or fully submerged objects, including living tissue. Broader impacts. Adhesives modeled after the sandcastle worm adhesive are likely to have widespread practical utility. For example, it may be possible to develop non-toxic, waterborne paints that can be applied to surfaces under water. The biggest impact of the biomimetic sandcastle glue may be in medicine. There remains much room for improvement in non-toxic adhesives that do not cause severe immune reactions that can be used on wet tissues damaged by accident or as a result of surgery. To summarize the findings, the sandcastle worm glue is naturally packaged in two types of pre-organized micron-sized granules. Each type of granule contains a distinct set of oppositely charged polyelectrolytes. When secreted, the bioadhesive is initially fluid but rapidly insolubilizes when the components of the two granule types interact and are exposed to seawater. Both granule types are pre-loaded with an inactive enzyme that becomes active after secretion to crosslink the fluid into a solid glue. The end result is tough and resilient underwater bonds formed with an energy-absorbing, water-filled adhesive foam. Like manmade bubble wrap used to protect delicate cargoes, the bubbles of the adhesive could deform at low loads, or rupture and collapse at higher loads, to dampen the peak load on the joint by dissipating impact energy. At high strain rates, pressure induced flux of water through open pores in the foam would provide additional viscous dissipation of impact energy. Additional studies of the sandcastle worm's glue could shed light on design principles for developing fluid-filled foam adhesives underwater from water-soluble components.
