Introduction to Biological Adhesion Biomimetics 2004 1997 1 1983a b Table 1. Research targets for biomaterial development Biological Target Description Keratin A hard, durable insoluble, structural protein that is the primary component of horns, hoofs, feathers, skin, hair, and nails; a scleroprotein Elastin An insoluble protein found in connective tissue and known for its elasticity and similarity to collagen; a scleroprotein Collagen A tough, insoluble, inelastic protein with high tensile strength that serves as the support structure in skin, tendons, and bone; a scleroprotein Silks High tensile strength protein fibers that contain various proteins (fibroin, spidroin); most commonly from spiders and silkworms Fibrin (and other coagulation system proteins) A sticky, insoluble, clot-forming protein formed by constituents in the blood; a scleroprotein Chitin A specialized carbohydrate containing nitrogen (nitrogenous polysaccharide); found in the cell walls of certain fungi and in the exoskeletons of arthropods Cellulose An insoluble complex carbohydrate (polysaccharide composed of linked glucose units); main constituent of the cell walls of plants Mucin A nitrogenous, conjugated protein (protein linked to a sugar) found in mucous secretions; acts as a lubricant and protects body surfaces The production of an underwater adhesive that mimics the properties of marine mussels is a challenge that has received considerable attention. Many of the mussel adhesive proteins identified to date are polyphenolic proteins. Polyphenolic proteins are nontoxic, biodegradable, and have low immunogenic qualities that make them highly attractive for environmental, medical, and industrial purposes. Biological Adhesion Naturally produced adhesives are common in many biological systems and are known for their superior strength and durability compared with man-made materials. Examples of specialized biological systems that generate a vast amount of adhesives research include bacteria, spiders, marine tubeworms, sea cucumbers, barnacles, and mussels. Many bacteria synthesize exopolysaccharides—extracellular protective adhesive matrixes. Caulobacter crescentus 2006 2001 2000 2003 Phragmatopoma californica 2004 2005 Holothuria forskali 2003 Mytilus spp 2005 2000 2005 2000 1997 M. edulis M. edulis Mytilus edulis M. edulis 1 M. edulis Figure 1 M. edulis a b c  Adhesion Mechanisms in Mussels Geukensia demissa Mytilus edulis Bathymodiolus childressi Modiulus modiolus 2006 1990 1998 1999 3+ 3+ 2004 2005 Mytilus californianus 2006 2003 1999 o 2 M. edulis Figure 2 M. edulis  2000 2002 o 2004 2006 2006 Components of Mussel Adhesion Attachment in Mussels: The Byssus 1952 1970 1985 2002 1952 1998 1995 Mytilus edulis 1985 M. edulis 3 M. edulis 4 M. edulis Figure 3 M. edulis  Figure 4 M. edulis  Mechanical Properties of Mussels M. edulis 6 −3 6 −3 6 −3 6 −3 6 −3 1988 6 −2 1999 1998 1979 1997 1996 1977 1977 M. edulis Byssal Thread Proteins Byssal Thread Polyphenolic Protein: Mefp-1 1981 1983b 1990 1983b 2005 Mytilus M. galloprovincialis 1994 M. coruscus 1996b M. trossulus 1995b M. californianus 1986 M. chilensis 1990 M 2004 Mytilus Dreissena polymorpha 1993 2000 Dreissena bugensis 2002 Perna viridis 2004 Perna canaliculus 2005 Guekensia demissa 1989 Limnoperna fortunei 1999 Aulacomya ater 1991 Choromytilus chorus 1990 1991 3+ 3+ 2002 Mytilus edulis 2006a Byssal Thread Polyphenol Oxidase http://www.chem.qmul.ac.uk/iubmb/enzyme/ o 1993 o o 1990 o M. edulis M. edulis 1981 1985 1996 M. edulis o 2000 M. edulis 1985 1996 2000 Byssal Thread Collagens n M. edulis 1979 1997 M. edulis M. galloprovincialis 2002 2004 Proximal Collagen (Col-P) 1997 1995 M. edulis 2+ 2000 1998 1996 Distal Collagen (Col-D) 1998 Pepsin-resistant Nongradient Collagen (Col-NG) 1998 n m M. edulis Proximal Thread Matrix Protein (PTMP) 2002 M. edulis M. galloprovincialis Byssal Plaque Polyphenolic Proteins Mefp-2 1995a 1992 2006b M. galloprovincialis 1995b M. coruscus 2000 D. polymorpha 1993 2004 Phragmatopoma californica Mefp-3 1995 1999 1999 2000 2006 M. galloprovincialis 1996a M. californianus 2006 Mefp-4 1998 1998 1999 M. californianus 2006a Mefp-5 Mytilus edulis 2001 M. galloprovincialis 2004 M. californianus 2006b Discovery of Additional Foot Proteins: Mcfp-6 2006b Adhesive Testing of Mussel Proteins A wide variety of adhesive tests have been applied to intact byssal threads, plaque, portions of threads, or materials bonded or coated with individual adhesive proteins. Gross comparative tests on byssal threads were described earlier in this review. 1985 1987 Attachment Failure 1985 Adhesive Techniques 2 Table 2. Examples of materials testing with mussel adhesive proteins or synthetic analogs containing repetitive motifs from mussel proteins Surfaces Tested Mussel Protein or Synthetic Analog References Slate Synthetic recombinant Mefp-1 1985 Silica (glass) Plastic acetal (acetate) Paraffin wax Polytetrafluoroethylene (PTFE) Polystyrene Recombinant Mefp-1 1990 Limestone/dolomite cobble D. bugensis D. polymorpha 1992 Mild steel Stainless steel Marine concrete 1995 Marine plywood Polyvinyl chloride Polymethylmethacrylate (Plexiglas®) 1996 Aluminum Teflon® D. bugensis D. polymorpha 1997 Concrete Mild steel Polyvinyl chloride Stainless steel Silicone Synthetic recombinant Mefp-1 1999 Silica Polyethylene terephthalate (PET) Teflon® Aluminum Synthetic polypeptide mimics of marine adhesives 1998 Steel Silica Plastics Microporous apatite surface Mefp-1 1998 Silica M. edulis M. californianus A. ater G. demissa 1999 Polytetrafluoroethylene (PTFE) Teflon® Nylon Iron Soda glass Methyl- and oligo (ethylene oxide)-terminated, self-assembled monolayers Mefp-1 (Cell-Tak™) and fibrinogen 2000 Germanium (oxide) Mefp-1, Mefp-2, and polylysine 2000 Polystyrene Poly octadecyl methacrylate 2001 Silica Mefp-1 2001 Porcine skin M. edulis 2003 Silica Recombinant Mgfp-5 and Mefp-1 (Cell-Tak™) 2004 Polymethylmethacrylate (Plexiglas®) Polystyrene Aluminum 2006 Modiolus modiolus Dreissena polymorpha Mytilis californianus M. edulis 2007 2006 As our ability to perform sensitive measurements with small amounts of protein improves, along with the increased resolution of the techniques used, we can anticipate that our understanding of the interactions of adhesive proteins necessary to achieve robust adhesion will increase. Future Supply of Adhesive Proteins by Recombinant Approaches Background M. edulis M. edulis M. edulis 1990 2007a Escherichia coli Saccharomyces cerevisiae Pichia pastoris Kluyveromyces lactis E. coli Promoter and transcriptional regulators are DNA sequences that direct gene expression. They can be native to the host, artificially added to the host DNA, or incorporated in vector systems. Translational stop signals can be incorporated into the DNA genes or are present on an expression vector. Prokaryotes and eukaryotes use different regulators for gene expression. Cultivation factors, such as energy sources, aeration (oxygen), temperature, and induction protocols are specific to the host organism or cell type and are dependent on the quantity used for production. Small-scale cultivation generally involves flasks or petri dishes and volumes less than 1 L. Larger-scale cultivation in the research laboratory setting can use bioreactors with volumes as large as 100 L. Automated monitoring and control of cultivation variables is used in large-scale recombinant protein production. M. edulis M. edulis 1990 1990 1999 The Recombinant Protein Approach S. cerevisiae 1991 2000 1994 2001 Mytilus 1990 S. cerevisiae 1993 E. coli 1999 E. coli A S. cerevisiae 2006a b M. galloprovincialis 1999 M. galloprovincialis M. galloprovincialis 1997 2004 E. coli 2005 E. coli 2007a b 2007a 2007b D. polymorpha 2000 E. coli D. polymorpha 2000 http://www.cnap.org.uk/ 2005 2007 Nephilla clavipes Current and Future Research Areas Related to Mussel Adhesion and Adhesives Commercialization of Mussel Adhesive Proteins Numerous economic factors are important in the production and synthesis of foreign proteins, whether using microbial cell culture, animal cell culture, plant tissue culture, transgenic plants, or transgenic animals. Production costs (yield for cost comparisons), safety issues (for therapeutic use), and stability of the product (the potential for the protein to degrade or lose function during extraction/purification procedures) are a few issues that require careful analysis before the method is chosen. Regulatory issues relevant to Good Manufacturing Practice (GMP) for production of therapeutic proteins must be followed. Any product containing recombinant mussel adhesive protein will require extensive testing and validation from health, environmental, and adhesives industries before commercialization. M. edulis Biofouling 1990 2001 2001 1999 2000 1997 1997 2003 2001 2006 2006 2005 Mussels from Extreme Environments 5 Bathymodiolus childressi 1996 M. edulis 5 M. edulis B. childressi M. edulis 5 2006 Figure 5 B. childressi M. edulis a B. childressi b B. childressi c M. edulis d M. edulis  Novel Applications for Mussel Adhesive Collagens and Polyphenol Oxidase 1998 2006 1996 M. edulis 2000 Medical and Dental Adhesives 2002 2006 2002 2002 2000 http://www.chem.ucla.edu/dept/Organic/garrell.html 2002 2003 1990 1988 1994 Conclusions Scientists and laymen alike have been fascinated for years with the ability of mussels to cling to surfaces under water. The ability to adhere in an aqueous environment, withstand numerous environmental forces, and resist conventional approaches to detachment are factors that continue to intrigue researchers today. During the last two decades, considerable time and effort has been spent in identifying the proteins that contribute to underwater adhesion by marine mussels. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to develop formulations for adhesives in which there exist endless applications for the commercialization of water-impervious, ecologically safe adhesives derived from mussels.