Marine-fouling invertebrates(e.g., mussel, barnacle, sandcastle worm, hydroid, and sea star) form a strong attachment to the marine substratum against mechanical stresses arising from the tide, buoyancy, and drag using their special physical and chemical underwater adhesives. There are many insightful lessons to learn from these organisms, and indeed, underwater adhesives from these marine organisms have been investigated as a source of potential underwater adhesives because of their fascinating properties. These properties include strong adhesion to various material substrates, water displacement, bioco- mpatibility, and controlled biode gradability. In addition, the marine environment has much in common with the human body; both systems are naturally saline and experience varied fluid flow, macromolecule-mediated fouling, and degradation of organic constituents via cell-level activities. These are just a few examples of matching biological and mechanical events present in both the human body and marine environment. Therefore, an understanding of how adhesives are produced by marine organisms will inspire new paradigms for the design and engineering of adhesives for medical use.
The mussel is one of the best model systems to understand how marine organisms anchor to the marine substratum effectively, due to lots of advanced researches on their biochemistry, physics and mechanics. Using fundamental research on mussel adhesion, there have been many attempts to translate insights from mussel adhesion to biomedical materials.
[Attachment of mussel to other mussel using byssal threads and schematic illustration of a byssal thread and adhesive plaque]
The mussel fabricates and utilizes what is called a byssus, which is a bundle of threads used to adhere the organism to the substratum. The individual thread is called a byssal thread. At the end of individual byssal thread, there is an adhesive plaque where underwater adhesion between the byssal thread and substratum occurs. Byssal thread is composed primarily of proteins and some trace levels of carbohydrates, lipids, and metal ions. Therefore, most research on mussel adhesion has focused on the adhesive proteins. Byssal threads are synthesized by the mussel foot, an extendable tongue-like organ, in which the protein precursors that compose the byssal thread are stored prior to thread formation. This is the reason why mussel foot protein is often considered when researchers describe mussel adhesive proteins. When a mussel makes a new thread, its foot emerges from its shell, finds a favorite spot for anchoring and secretes the precursors for byssus into a groove running along the length of the ventral side of the foot. Muscular contractions of the foot around the groove follow and the precursors assemble as a thread within 10 min. A byssus is composed of approximately 25-30 different types of precursor proteins and 10 of them have been characterized in biochemical and molecular biological studies. The precursor proteins are classified as byssal prepolymerized collagens (PreCols), thread matrix proteins(tmps), and foot proteins(fps) based on their roles and biochemistries. PreCols and tmps comprise the byssal thread filler and matrix, respectively, whereas fps is found in the adhesive plaque and acts an adhesive pad for underwater adhesion. To date, 6 types of fps have been isolated from the Mytilus genus, and 3,4-dihydroxyphenylalanine(DOPA), a hydroxylated tyrosine, due to post-translational modification of its benzene ring, is found in all of the identified fps, which implies that it may play an important role in mussel adhesion and byssus assembly.
Production capability and superior properties are very important for practical applications of mussel adhesive proteins. The natural proteins were initially extracted to study their biochemical properties; however, limited productivity hampered investigation of bulk-scale adhesive properties and biomedical and industrial applications. Thus far, extracts of mussel adhesive proteins containing primarily fp-1 and fp-2 are used commercially as a cell and tissue adhesive. Due to highly limited production processes, natural extraction appears unfeasible for bulk-scale practical applications of the protein, although the adhesive properties, such as versatile adhesion in wet environments, biodeg- radability and biocompatibility, have come to be recognized as very attractive. Recombinant DNA technology has been considered as a solution for obtaining large amounts of adhesive proteins for bulk-scale adhesion and practical applications. Recombinant protein expression systems have been used in various host cells, including bacterial, yeast, insect, plant, and mammalian cells, and the technology has greatly improved for mass-production of target proteins. cDNAs of mussel adhesive proteins were introduced heterologously into various vector systems and their over-expression has been attempted, with partial success. Synthetic constructs of the mussel adhesive components have also been prepared as an alternative approach for obtaining large amounts of adhesive proteins, and the initial low production and purification yields have now been greatly improved and the resulting proteins have been applied practically.
[Bulk-scale production of recombinant mussel adhesive proteins]
Ultimately, we expect that mussel adhesive proteins can be used as bioadhesives for binding items together in dry and wet environments, given their bulk-scale adhesive properties and biocom- patibility. Moreover, mussel adhesive proteins will be utilized in various biomedical and industrial fields in the near future, given their diverse adhesive properties even in wet conditions, enviro- nmentally friendly properties and high biocompatibility and high productivity and economical cost. Recent results have shown the high potential of adhesive biomaterials as cell and tissue adhesives and extracellular matrix and scaffold-coating materials for tissue engineering, immobilizing agents for biochip preparation, gene and drug delivery carriers and skin and bone adhesives.
Recently, a layer-by-layer technology using mussel adhesive proteins and glycosaminoglycans (GAGs) was deve- loped, where GAGs, such as hyaluronic acid, heparin sulfate, chondroitin sulfate and dermatan sulfate, were used for many cell- signaling events. GAGs were simply coated onto various surfaces using recombinant mussel adhesive proteins and enhanced cellular behavior was observed on the functionalized surfaces. The results suggested that negatively charged ECM molecules and ECM peptides could be efficiently immobilized and utilized in various tissue engineering and medical implantation applications. Moreover, novel, functional nanofibrous scaffolds based on recombinant mussel adhesive proteins were fabricated, which provided a mechanically durable structural backbone with the functionality of bioactive peptides. Moreover, facile functionalization of the nanofiber surfaces with various biomolecules was achieved using the adhesive and charged properties of mussel adhesive proteins, without surface modifications. These two- and three-dimensional strategies and the aforementioned cell adhesion abilities of mussel adhesive proteins have greatly accelerated the practical use of mussel adhesive proteins for the cell adhesion and tissue engineering fields.
[Facile surface functionalization of ECM molecules using mussel adhesive proteins]
[Reinforced multifunctional nanofibrous scaffolds using mussel adhesive proteins]
The BC domain of protein A(antibody-binding protein; used as an antibody immobilizing linker) was fused with the N-termini of fp-5, which was overexpressed satisfactorily in E. coli. The fp-5 of the fusion protein enabled direct coating, without any modifications, onto diverse surfaces including glass, polymers and metals, and the BC-domain showed excellent antibody-binding to the surfaces. This new strategy for effective and simple immobilization of antibodies onto diverse unmodified surfaces showed the high potential of mussel-adhesive proteins as linking materials and can be successfully applied to prepare biochip surfaces permitting antigen-antibody interactions. It is expected that similar strategies may be utilized to efficiently immobilize other biomolecules onto various surfaces.
[Schematic representation of simple and efficient antibody immobilization on diverse surfaces coated with BC-MAP]
In addition, the potential use of fp-151 as a gene delivery material was investigated, in view of its similar basic amino acid composition to histone proteins. fp-151 exhibited efficient DNA binding ability and transfection efficiency in mammalian cells, which demonstrates the potential of using mussel adhesive proteins as gene delivery carriers. Complex coacervates of mussel adhesive proteins have also been used in microencapsulation to initially protect and subsequently release encapsulated biol gically active compounds, such as hyd- rophobic drugs and food ingredients. As a model of microencapsulation, red pepper seed oil was used due to the ease of monitoring the encapsulation using fluor- escence, and interfacial coacervations of approximately 1-30 μm in diameter formed spontaneously. These results suggest that the microencapsulation system could be a useful component of the development of new adhesive biomaterials, including self-adhesive microencapsulated drug carriers.
Marine mussels attach rapidly to various solid surfaces in wave-swept seashores. The superior properties of the adhesives, such as strong adhesion with flexibility, bioco-mpatibility, and biodegradability, have resulted in their consideration as very promising biomaterials. Isolation of the mussel adhesive protein and biochemical studies have greatly increased the potential for their use as the next generation of biomaterials, and bulk-scale productions have accelerated practical applications, especially in the biomedical and tissue engineering fields. In particular, bioadhesive preparation and formulation for multi-functional biomaterials will be enabled by recent technological improvements in production of recombinant synthetic constructs from mussel adhesive proteins. This strategy will be expanded to discover new biomaterial and mimic natural materials successfully.