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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products Session 3: Biomaterials and Bioengineering THE COMMERCIALIZATION OF A BIOPOLYMER EXTRACTED FROM THE MARINE MUSSEL, MYTILUS EDULIS Christine Benedict Director, Quality Assurance Geneva Pharmaceuticals, Inc. In turbulent intertidal zones throughout the world, such marine animals as barnacles and mussels tenaciously attach themselves to slippery wet rocks, pilings, or any solid surface. The adhesives they use are remarkably effective. Scientists have researched these natural adhesives with a vision toward the development of new products for applications in science, medicine, and industry. Marine biologist J. Herbert Waite discovered the active ingredient of the mussel adhesive. He named it “polyphenolic protein.” This molecule forms strong, flexible bonds on virtually any surface, natural or manufactured, wet or dry. The secret of its versatility is the protein’s unusual chemical composition. Not only does it readily interact with manufactured materials, such as metals and plastics, but it also bonds to living tissue, such as bone and skin. Because it is a protein, the adhesive is gradually broken down by natural processes within the human body. This property of resorbability, together with a setting time that can be adjusted
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products as needed for specific uses, suggests that the polyphenolic protein could be the key component in an effective surgical adhesive. BioPolymers’ strategy was to commercialize specially designed adhesive formulations through licensing, cooperative development programs, and joint ventures with companies in the targeted markets. Customers in biomedical research have been using the adhesive to simplify the manipulation of cells and tissue outside the body. BioPolymers, Inc. spent 5 years focused on developing and commercializing novel adhesives and coatings for the medical and industrial markets. That concept was spawned in 1985 after Dr. Waite’s discovery of a decapeptide (sequence of 10 amino acids) which repeated 85 times to comprise the mussel adhesive protein (MAP). After licensing this technology from the University of Connecticut (patents 4,585,585 and 4,687,740), the company selected the patented 10 amino-acid sequence and variations of it for commercial development, although it was not known at the time which amino acid in that sequence was the proper starting point for the decapeptide. The mussel in its sea environment adheres to many substrates, and laboratory research indicated that extracted and synthetic protein polymers could stick to almost any substance, such as rock, plastic, metal, glass, Teflon, and skin. Even though the original concept was never demonstrated to work as an adhesive or coating in a medical or industrial environment, the fact that the mussel anchors itself by a collagen byssus thread to these surfaces supported the notion that the MAP could also adhere to the collagen of biological tissue. Proof of that theory was part of the responsibility of the new company. During its first 2 years, BioPolymers extracted the natural MAP and developed a product, CELL-TAK, to enhance cell attachment and, where appropriate, cell growth, in laboratory culture dishes. However, the market was small, and financial return too limited to warrant further expansion in this area. Hence, a distribution agreement was signed with another cell-culture product company. Also, during the first 2 years, the company’s attention focused on the medical field. A patent application was filed covering a myriad of end uses for formulation of mussel-based adhesives. In 1986, BioPolymers also conducted gross feasibility tests of the MAP in several ophthalmological in vitro and in vivo models. Some were successful, but recognizing the limitations in the supply of mussels and the degree of difficulty in moving a natural material through the FDA’s regulatory approval process, a significant research and development emphasis was
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products to focus on developing a synthetic version of MAP. The initial breakthrough was accomplished in late 1987 by attaching the chemically synthesized decapeptide to a backbone to form a copolymer with high molecular weight similar to natural MAP. By mid-1988, a cross-linking agent was found, and when combined with the synthetic MAP, appeared stronger than cross-linked natural MAP in in vitro studies on attaching skin to skin and cornea to cornea. By the fall of 1988, short-term experiments to develop specific formulations for wound management (the largest medical market opportunity) began in vivo in pigs. Results were very encouraging; animal shear strengths were more than sufficient to hold a skin graft in place over its entire surface area. When testing was expanded beyond the initial period, the experiments failed; strengths were at or below the level of controls. In drier, lower stress biological systems, such as the eye, the adhesive bond survived significantly longer for closing corneal perforations in rabbits and for corneal-type transplants in primates. Unfortunately, neither bond was sufficiently long enough to complete the healing process. BioPolymers entered 1989 focused on reformulating the adhesive components and retesting the combinations with the assumptions that the original “ingredients” were correct, but the “mix” might have been wrong for the in vivo environment. Continued experimentation, scientific analysis, and consultation with several expert scientists in related fields failed to pinpoint the exact cause of failure. Before it could expect success with a synthetic version, the evidence and collective opinion suggested that BioPolymers had to complete its understanding of how the mussel attached to objects and, in particular, to identify other components that work with the natural MAP in the adhesion and coating process. Primary Research Areas Basic Mussel Research Although the decapeptide was originally identified as the functional component of the adhesive, it is now believed that other components are also necessary. Working with Dr. Waite at the University of Delaware and researchers at the University of Connecticut Health Center, BioPolymers undertook the process of isolating and identifying the other components. Perhaps these “missing” components could better utilize DOPA, the amino acid in the protein, in in vivo animal studies. BioPolymers’ synthetic and natural MAP formulations have exhib-
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products ited acceptable shear strength, but consultants in adhesive technology reported that an adhesive must also have peel strength. Although the mussel’s adhesive in nature is observed to have peel strength, current formulations, as measured in the laboratory, do not. This may be further confirmation that components to bind with MAP are missing. Conformation of the Decapeptide An outside effort at the University of Illinois found that the decapeptide on the backbone, as it was synthesized, may not be able to fold into the correct shape as natural MAP to attach to the collagen in skin or bone. More amino acids may be needed in the peptide—not 9 or 10 as it was designed. There were indications that the decapeptide should have started with a different amino acid in the chain to provide the proper shape to integrate with collagen. Novel Polymer Synthesis and Formulation Development For the reasons cited above as well as other characteristics of the synthetic MAP, the limited successes achieved with the synthetic MAP did not effectively utilize DOPA—the proprietary technology—in the curing process. Formulation research should include wound bed stabilizers, as are found in other tissue adhesives, such as fibrin. If these technical tissues were resolved, new components identified and utilized in formulations, the research and development effort could move quickly into testing, because the comprehensive in vitro, in vivo, and safety models developed are still applicable and relevant. The safety and efficacy testing protocols were considered to be significant assets of BioPolymers. The water-based, proteinaceous polymer adhesive system being developed at BioPolymers required significant adaptation and test development to achieve reliability and reproducibility in results. A large investment in research time and consultation by adhesion test experts resulted in well-defined protocols for in vitro and in vivo tests. With further research, new formulations and modified polymer or cross-linking systems could readily be evaluated against a large body of data using the established test systems.
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products Industrial Applications Technology Review Status In 1990, a research and development project to investigate the industrial applicability of MAP was initiated in the areas of anticorrosion coatings and metal sequestering (for contaminated wastewater and metal reclamation). Early experiments focused on the development of protocols, model systems, and initial feasibility studies. Specialty Coatings To test the capacity of DOPA-containing polymeric systems to protect metals against corrosion, development was underway to establish techniques for cleaning surfaces of metals, such as copper, carbon-steel, and aluminum. Because cost is the driving force in the protective coatings area, a search of the literature for inexpensive, readily available, water-soluble polymeric substances that could be converted to DOPA-containing coating systems was completed. Several polymers were identified and toll vendors were being sought. After these fundamental anticorrosion data were acquired, the plan was to use a number of specialty ink and coating formulator consultants. Metal Sequestering Because of the strategic nature of certain rare metals required in hightech military manufacturing procedures, protocols for the sequestering of Group VI metals from solution were being developed. Some of the metals targeted for chelation included tungsten, molybdenum, chromium, and vanadium. Early experimental results were very promising. A 200-fold decrease in concentration from a solution of these metals was accomplished with supported heterophase DOPA and DOPA analog complexing agents. Unfortunately, a cursory literature search of the catecholate metal complex patent revealed a large body of previous art. However, specific patent searches on supported catecholate complexing agents did not identify previous art that would preclude patent protection for this technology. Further, examination of supported polymeric sequestering agents that would fall beneath the umbrella of U.S. Patent 4,908,404 still needed to be con-
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products sidered. In addition, a great deal more experimental work was required to capitalize on the experimental advances already accomplished. Biopolymer Patents Issued Adhesives Derived from Bioadhesive Polyphenolic Proteins (No. 5015.677, 1991) This invention relates to adhesive formulations that are derived from bioadhesive polyphenolic proteins and used in a wide variety of applications. This claim currently covers a formulation that has a bioadhesive polyphenolic protein component with a specific weight percent of a proteinaceous substance comprising about 50–150 repeating units of the decapeptide and from about 1 to 40 percent of a cross-linking agent that promotes cross-linking of the decapeptide. The formulation optionally also includes additives that promote desired properties of the formulation and fillers. Synthetic Amino Acid and/or Peptide Containing Graft Copolymers (No. 4,908,404, 1990) This invention relates to a peptide-containing graft copolymer with a molecular weight of about 30,000 to 500,000. It includes a polymer backbone containing or capable of modification to include free primary or secondary amine functional groups for reaction with an amino acid or peptide graft and an amino acid or peptide graft reacted with at least 5 percent to 100 percent of the primary or secondary amine groups on the polymeric backbone. The amino acid or peptide graft comprises at least one dihydroxyphenylalanine (DOPA amino acid) or a precursor thereof capable of hydroxylation to the DOPA form.
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products SELF-CLEANING SURFACES: BIOLUBRICANTS, DRAG REDUCTION Anne E. Meyer, Ph.D. Principal Research Scientist, Biomaterials State University of New York at Buffalo Imagine yourself cruising in a sleek, new fishing boat off the Atlantic shore, making good speed, when suddenly porpoises, so common in these waters, begin to frolic near your boat. Although you are going as fast as you can, these friendly marine mammals probably will not only keep up with you, but will swim circles around you, even without riding your bow wave. After some six months in the water, you will find that your vessel will not be able to make anywhere near the top speed it did when it was freshly launched with a new coat of antifouling paint. However, the porpoises and their cousins, the killer whales, who will have been in the water much longer than your boat, will have no trouble maintaining their speed. (Baier and Meyer, 1986). The Marine Environment Marine mammals, fish, and other organisms have evolved to display different techniques to reduce drag and increase “lubrication.” Many of these same organisms also remain relatively free of biological fouling throughout their lives or during critical stages of their development. Certainly, the sleek shapes of many of the swimming creatures contribute greatly to their ability to move through the water easily, while expending relatively little energy. Textural features also may play a role in the reduction of “form drag.” The circumferential ridges that dolphins can produce in their skin, for instance, may serve to increase the drag-reducing boundary layer around the quickly moving mammal. Research and development of synthetic forms and materials primarily has mimicked the macroscopic forms and textures of marine mammals and fish. This approach has been well exercised for more than a thousand years for the design and use of fishing and exploration vessels and, more recently, submarines, torpedoes, and ocean-drilling platforms. The study of the micro- and submicroscopic tools developed by nature for drag reduction and lubrication is a more recent phenomenon. At the microscopic and molecular level, characteristics of the water-contacting surfaces of low-drag and fouling-resistant marine organisms and plants fall
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products into two general categories: those that reduce drag and fouling by sacrificial slime, exudate films, or tethered macromolecules on their surfaces; and those that achieve these effects through the intrinsic low-surface-energy of their cellular and tissue surfaces. The development of fouling-release paints and coatings over the past 30 years has produced a marked reduction of drag along ships, primarily due to the significant decrease in hard fouling (e.g., barnacles and tubeworms) of these coatings. Is there, however, another significant increment of improvement that could be obtained from additional study of natural marine organisms and surfaces? And how can these findings be translated to the biomedical marketplace? The Biomedical Environment Although work on tethered polymers (e.g., polyethylene oxides) for biomedical applications has been extensive in recent years, no consistently satisfactory molecular-level approach to drag reduction and lubrication has been developed for long-term use. Perhaps answers rest with the confounding complexity of natural fluids and surfaces at the molecular level. Preliminary experiments have shown that proteins and other macromolecules in seawater and other biological fluids can form multicomponent films that have lower coefficients of friction than films formed from the same fluids under low shear conditions. Films formed from less complex fluids do not so easily reduce interfacial friction or drag. The report resulting from the October 1995 National Institutes of Health workshop titled “Biomaterials and Medical Implant Science: Present and Future Perspectives” outlined approximately 25 priorities for the design, development, and manufacturing of safe and effective medical devices (Watson, 1996). These priorities included: Biologically based materials—including “smart” materials for cell-based, drug-based, and gene-based therapies—designed by building biological structure and function into materials. Cross-disciplinary core infrastructures in research, design, and education. Development of strategies for synthesis and methods for generating new materials and coatings and development of new, alternative, or improved materials.
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products All these priorities present open invitations to marine scientists and marine natural products to enter the biomedical field. There continues to be a substantial need for self-cleaning materials and lubricating coatings in the biomedical environment. Typical applications include many types of implanted devices (e.g., catheters of all types, heart valves, and contact lenses) and extracorporeal devices (e.g., dialysis membranes and blood oxygenators), as well as palliative treatment for clinical conditions, such as Sjogren’s syndrome (dry eye and dry mouth). Flotsam, Jetsam, and Neuston In addition to harmful anthropogenic components at certain times, the sea surface is a treasure chest of organisms (including bacteria), surfactants, and proteins. Concentrations of materials in the uppermost micrometer and millimeter of the ocean’s surface (MacIntyre, 1974) are addressed in terms of “enrichment factors,” the ratios of microlayer concentrations to subsurface concentrations. A key function of the sea-surface layer is control of gas and chemical exchange between the atmosphere and the water (Liss and Duce, 1997). Might there be additional lessons here for biomedical applications? Can marine surfactants and proteins provide technology for improved treatment of lung failure, renal failure, or acute liver failure? Is there a biocompatible, oxygen-carrying marine material from neuston or plankton that could serve as an artificial blood? And, getting back to the eyes and mouth, can Sjogren’s syndrome be overcome by a protein or surfactant from the sea surface that would lubricate and allow gas exchange and retard evaporation from natural mucosal surfaces? What are the qualities of killer whale “tear gel” in this regard? Marine Product Development: “Assistive Technologies” Although the focus of this workshop is on biomedical applications of natural marine products, we also should consider how these organisms and molecules will be renewably collected from marine life or mined from the sea surface, the subsurface, and the sea floor. Selection of suitable materials and coatings for sea surface or underwater processing facilities will be critical to minimize environmental impact and to maximize process efficiency. Self-cleaning and drag-reducing materials also have a key role to play as
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products assistive technologies in the seeding, harvesting, and development of natural marine products. Surface-fouling periphyton and mussels, typically cited as troublesome invasive biofoulers, have high productivity and filtration capacities that offer promise for biotechnological product processing and water-quality management. Designs have been presented that use the bioadhesive potential of natural foulers for flow-through mussel filters to clear bioavailable contamination from effluents before discharge (Diggins et al., 2002). Scale-up of these designs for treatment and collection of trace marine natural products could be a practical path to harvesting of new medicinal agents. Summary The usual trend in fields of science and engineering is to replace precious natural products with synthetic substances. In at least one case, however, a natural marine product had no substitute: some fish oils were so effective as lubricants in jewel-bearing watches and highly sensitive gyroscopes, that no synthetic product was ever found to replace them. Instead, digital watches replaced the mechanical instruments and synthetic fluoropolymers were developed to contain (but not yet replace) the natural lubricants from the sea. Until we know otherwise, we must assume that the marine environment continues to hold many molecules of great potential for lubricants, self-cleaning surfaces, and molecular-exchange films. The following references provide some of the context for links between marine science and biomedical engineering. References Baier, R. E., and A. E. Meyer. 1986. Biosurface chemistry for fun and profit. Chemtech 16:178-185. Diggins, T. P., R. E. Baier, A. E. Meyer, and R. L. Forsberg. 2002. Potential for selective, controlled biofouling by Dreissena species to intercept pollutants from industrial effluents. Biofouling 18:29-36. Liss, P. S., and R. A. Duce (eds.). 1997. The Sea Surface and Global Change. Cambridge University Press. MacIntyre, F. 1974. The top millimeter of the ocean. Scientific American 230:62-77. Watson, J. T. 1996. Biomaterials and medical implant science: present and future perspectives: a summary report. Journal of Biomedical Materials Research 32:143-147.
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products UNIFORM MICROPOROUS BIOMATERIALS PREPARED FROM MARINE SKELETAL PRECURSORS Rodney A. White, M.D. Professor, Vascular Surgery Harbor-UCLA Medical Center and Eugene W. White, Ph.D. X-ray Analytical, Inc. Highly interconnected microporous materials are difficult, if not impossible, to produce synthetically. With the aid of marine life-forms, we are now able to fabricate materials with desirable characteristics. Replamineform, meaning replicated life-forms, describes a method for making microporous materials made by using the calcium carbonate skeleton of several forms of marine life as a template (White et al., 1972). Three-dimensionally microporous skeletons are found in echinoderms and certain species of coral. The sizes of pores are uniform and range from 15 to 500 micrometers (µm), depending on the species. Replicas of the microporous skeletal framework are prepared by investing the calcium carbonate skeleton with metals, ceramics, or polymers and then removing the calcium carbonate with a mild acid solution. The residual material is an interconnected porous structure. Precursor skeletons and the composite structures can be easily shaped to desired configurations prior to or after casting. Replamineform materials have many applications; to date, they have been primarily evaluated as medical devices and prostheses. The size of the microporosity makes the process ideal for making artificial organs and implants that become ingrown with host tissues. Microporous biomaterials can be used to replace bone, blood vessels, trachea, and other damaged organs and tissues. High-surface-area membranes and piezoelectric-pyroelectric composites have also been described using this technology. Replamineform Process Replamineform biomaterials are fabricated using the calcium carbonate skeleton of certain forms of invertebrate marine life as a template. Echinoderms have a three-dimensional calcite lattice, topographically known as
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products a periodic minimal surface (Donnay and Paulson, 1969). Such surface divides spaces into two interpenetrating regions, each of which is a single, interconnected domain. The interface between the solid calcite phase and the organic material of the animal provides maximal surface area. A similar structure is found in the aragonite skeletons of some perforate reef-building corals. The skeletal microstructure of the colonial coral Porites, for example, has a high degree of uniformity of pore diameter and a solid-to-void ratio of approximately one. Exceptionally high permeability is achieved because every pore in the meshwork is connected to all other pores (Weber and White, 1973). Positive or negative replications of the microstructure can be produced using direct impregnation or lost wax casting techniques (Weber et al., 1971; Weber and White, 1973). The calcium carbonate template is removed by immersing the composite in dilute acid solution. Precursor skeletal materials are easily shaped to the desired configuration prior to acid etching. Biomedical-grade elastomeric polymers are most promising in this application. Replacement of the porous calcium carbonate of corals (aragonite) and echinoids (calcite) by hydrothermal conversion with ammonium dibasic phosphate has been achieved (Roy and Linnehan, 1974). Virtually complete conversion to the calcium phosphate derivative (hydroxyapatite or whitlockite) with retention of the original microporous structure has been reliably accomplished. Biomedical Applications Replamineform biomaterials are under investigation in several applications. A brief overview of some of the medical applications is presented. Hard Tissue Prostheses The well-known capacity of bone tissue to regenerate has prompted considerable enthusiasm into its research. One area of interest is the interfacing of bone with prostheses to form permanent attachments without relying on adhesives. Replamineform microporous ceramics (alumina and titanium) and metals (vitallium) have been implanted up to 8 weeks in the cancellous bone in the lower extremity of the mongrel dog. New bone was found to grow into the pores of the materials and become mineralized (Chiroff et al., 1975). Dunn et al. (1979) reported favorable results using
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products segmental femoral prostheses. The implants were cast as a solid core of Zimalloy with a replamineform 300-500 µm porous coating. There was no evidence of infection, and 10 of 11 dogs were alive and ambulatory without difficulty at 17-19 months post-surgery. Another area of interest to orthopedic surgeons is the regeneration of bone without permanent prostheses. Replamineform hydroxyapatite is being investigated as a long-term biodegradable bone substitute. It has been shown to provide a lattice through which bone regeneration occurs (Holmes, 1979). Hydroxyapatite implants have been evaluated in dogs as replacements in long-bone and mandibular discontinuities, as onlay bone grafts (frontal sinus and manidibular subperiosteal) and gluteal muscle implants (Roser et al., 1977). These studies demonstrated that hydroxyapatite implants can be custom-shaped intraoperatively, are well-tolerated by host tissues, provide a scaffolding for the ingrowth of bone and connective tissue, and do not induce bone regeneration when implanted in soft tissue. Cardiovascular Materials Successful replacement of diseased arteries and veins is a challenge to modern medicine. Small internal diameter synthetic prostheses have not functioned well for several reasons. First, no surface has been developed that is passive to the body’s clotting mechanisms. Second, normal blood vessels have compliance properties (i.e., they expand with each pulsation), which are much greater than those of currently available prosthetic grafts (Kidson and Abbott, 1978). If there is a mismatch between the compliance of the prosthesis and the host’s blood vessel, turbulence and stresses are generated within the graft. Finally, prosthetic vascular grafts do not have the porosity required (Harrison, 1961; Wesolowski, 1962). Porosity of prosthetics allows for incorporation of surrounding fibrous tissues on the outside of the graft and regeneration of a viable neointimal surface on the inside. Our research program addresses each of these deficiencies. Replamineform vascular prostheses fabricated in medical-grade polyurethane become rapidly incorporated with a thin, stable, neointimal flow surface (White et al., 1976). Similar results have been generated using vascular grafts made of silicone rubber, and in fact, independent effects of pore size and biomaterial on tissue incorporation of the prostheses have been described (Hiratzka, 1979). Work in the laboratory of the investigators has revealed a greater than 90 percent patency rate for 4-mm I.D.,
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products 6-cm-long silicone rubber prosthetics. Current work shows the efficacy of matching the compliance of the prostheses to the native artery. Tracheal Prostheses Tracheal obstruction as a result of malignant compression or trauma frequently requires resection and reconstruction of the involved segment. Replacement of the trachea with a prosthesis is difficult for the experimental surgeon, because this tubular organ functions in an environment continually contaminated by microbes. Thus, tissue incorporation of the prostheses may be inhibited by chronic infection. Early work demonstrated successful replacement for up to 21 months in mongrel dogs using 3-cm-long replamineform microporous tracheas (Nelson et al., 1979). Bioelectric polyurethane prostheses with a pore range of 120 to 180 µm appears to provide a favorable lattice for tissue incorporation of the prosthetic wall. Potential Industrial Applications High-surface-area membranes and piezoelectric-pyroelectric composites have been described using this technology. The potential applications of these materials have not been explored beyond early feasibility. References Chiroff, T. T., R. A. White, J. N. Weber, and D. M. Roy. 1975. Tissue ingrowth of replamineform implants. Journal of Biomedical Material Research Symposium 6:29-45. Donnay, G., and D. L. Paulson. 1969. X-ray diffraction studies of echinoderm plates. Science 166:1147-1152. Dunn, E., T. Brooks, B. Gordon, S. Rothert, E. White, and L. Tarhay. 1979. Replacement of the canine femoral diaphysis with a porous coated prosthesis. Johns Hopkins Medical Journal 145:101-106. Harrison, J. H. 1961. Influence of porosity on synthetic grafts. Archives of Surgery 82:8-18. Hiratzka, L. F., J. A. Goeken, R. A. White, and C. B. Wright. 1979. In vivo comparison of replamineform Silastic and bioelectric polyurethane arterial grafts. Archives of Surgery 114:698-702. Holmes, R. E. 1979. Bone regeneration within a coralline hydroxyapatite implant. Plastic and Reconstructive Surgery 63:626-633. Kidson, I. C., and W. M. Abbott. 1978. Low compliance and arterial graft occlusion. Circulation 58:I-4, I-9. Nelson, R. J., R. A. White, R. S. Lawrence, F. M. Hirose, and M. D. Walkinshaw. 1979.
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products Development of a microporous tracheal prosthesis. Trans American Society of Artificial Internal Organs 25:8-12. Roser, S. M., F. A. Brady, and B. McKelvy. 1977. Tissue ingrowth of hydroxyapatite replamineform implants in the dog. Paper presented at American Association of Dental Research Symposium, Las Vegas, Nev. Roy, D. M., and S. K. Linnehan. 1974. Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange. Nature 247:220-222. Weber, J. N., and E. W. White. 1973. Carbonate minerals as precursors of new ceramic, metal, and polymer materials for biomedical applications. Mineral Science and Engineering 5:151-165. Weber, J. N., E. W. White, and J. Labiedzik. 1971. New porous materials by replication of echinoderm skeletal microstructures. Nature 233:337-339. Wesolowski, S. A. 1962. Evaluation of tissue and prosthetic vascular grafts. Chas C. Thomas, Inc., Springfield, Ill. White, R. A., F. M. Hirose, R. W. Sproat, R. S. Lawrence, and R. J. Nelson. 1981. Histopathologic observations after short term implantation of two porous elastomers. Biomaterials 2:171-176. White, R. A., J. N. Weber, and E. W. White. 1972. Replamineform: a new process for preparing porous ceramic, metal and polymer prosthetic materials. Science 176:922-924. White, R. A., E. W. White, E. L. Hanson, R. F. Rohner, and W. R. Webb. 1976. Preliminary report: evaluation of tissue ingrowth into experimental replamineform vascular prostheses. Surgery 79:229-232. BIOMATERIALS FOR TISSUE ENGINEERING, DRUG DELIVERY, AND OTHER MEDICALLY RELATED APPLICATIONS: THE MARINE SOURCE Cato T. Laurencin, M.D., Ph.D. Helen I. Moorehead Professor of Chemical Engineering Drexel University Clinical Professor of Orthopaedic Surgery Research Professor of Pharmacology and Physiology MCP-Hahnemann School of Medicine Emerging interest in the use of biomaterials derived from marine sources has led to a variety of new approaches for the treatment of disease. Homopolymers and copolymers based on hydroxybutyrate and hydroxyvalerate are bacteria-derived aliphatic polyesters that exhibit thermoplastic properties. A number of bacteria, most notably Alcaligenes eutrophus, produce these materials as a carbon reserve under certain condi-
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products tions. As a biomaterial, they have the practical advantages of being inexpensive and readily produced through fermentation techniques. Their biocompatibility has been studied in a number of settings and is similar to more conventional polymer-based systems used clinically. Degradation of these polymers takes place through surface-erosion mechanisms; their degradation is ostensibly dependent on enzymatic mechanisms with slow hydrolytic degradation. In tissue engineering, these polymers have been proposed for use as possible bone substitutes with Jiang et al. (2001), who described the development of new composites of poly(hydroxyalkanoates) and hydroxyapatite. In preliminary studies, these composites exhibited tensile strength and moduli comparable to cancellous bone. Other new work has centered on antibiotic delivery for the treatment of periodontal and other diseases. Hydroxyapatite materials derived from coral can be formulated to allow the attachment and growth of cells. Such factors as macroporosity and microporosity and degree of degradability are important determinants in cellular and tissue response and clinical outcomes. Early studies by Laurencin et al. (1996) identified the ability of coralline materials to permit attachment and growth of osteoblasts with maintenance of phenotypic expression in these cells. In bone-tissue engineering, these hydroxyapatite materials have been used as parts of composites for hard-tissue regeneration. The hydroxyapatite acts as a reinforcing phase for the matrix and modulates mechanical properties while permitting the maintenance of biological response. This work has led to the development of a family of three-dimensional matrix forms for bone-tissue engineering that serve as templates for bone repair and regeneration. Chitosan is a marine-derived polysaccharide material that has received increasing interest in biomedical applications. The material appears to exhibit little local or systemic toxicity at implantation, is sterilizable through various methods (such as autoclaving), and can be processed in a variety of distinct ways. The material demonstrates special properties, such as the formation of colloidal particles that can form complexes with macromolecules. Its ability to carry out this process in conjunction with DNA offers delivery-vehicle methods often at the nanolevel. In delivery of drugs ranging from antibiotics to various anticancer species, the polysaccharide is able to be utilized as a vehicle for delivery through oral, nasal, and parenteral routes. Chitosan has shown surprising affinity for a number of mesenchymal-derived cells, such as chondrocytes and osteoblasts, and thus may have important applications as part of tissue-engineered musculoskeletal matrix
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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products systems. For bone and cartilage repair, work has begun to explore the development of polymer-chitosan matrices for tissue- engineering and drug-delivery applications using microsphere matrix technology previously applied to polymer-ceramic systems. It is hoped that this work will result in robust constructs that may be used in a variety of musculoskeletal environments. In summary, a heterogeneous group of marine biomaterials (organic polymer based, ceramic based, and polysaccharide based) present important alternatives and challenges for use in biomedical applications. Collectively, their true places in drug delivery, gene therapy, and tissue engineering is yet to be determined. Bibliography Dash, A. K., and G. C. Cudworth. 1998. Therapeutic applications of implantable drug delivery systems. Journal of Pharmacological and Toxicological Methods 40:1-12. Felt, O., P. Buri, and R. Gurny. 1998. Chitosan: A unique polysaccharide for drug delivery. Drug Development and Industrial Pharmacy 24:979-993. Janes, K. A., P. Calvo, and M. J. Alonso. 2001. Polysaccharide colloidal particles as delivery systems for macromolecules. Advanced Drug Delivery Reviews 2001 47:83-97. Janes, K. A., M. P. Fresneau, A. Marazuela, A. Fabra, and M. J. Alonso. 2001. Chitosan nanoparticles as delivery systems for Doxorubicin. Journal of Controlled Release 73:255-267. Jiang, T., and P. Hu. 2000. Surface modification of hydroxyapatite to introduce interfacial bonding with polyhydroxybutyrate in a biodegradable composite. Paper pesented at the 16th Annual Meeting of The Polymer Processing Society, 2000. Jiang, T., P. Hu, Y. Li, and L. Liu. Submitted. Development of polyhydroxyalkanoates-bioceramics: Nanocomposites for bone substitute. Journal of Tsinghua University. Laurencin, C. T., M. A. Attawia, H. E. Elgendy, and K. M. Herbert. 1996. Tissue engineered bone-regeneration using degradable polymers: the formation of mineralized matrices. Bone 19:93S-99S. Lee, Y., Y. Park, S. Lee, Y. Ku, S. Han, S. Choi, P. R. Klokkevoid, and C. Chung. 2000. Tissue engineered bone formation using chitosan/tricalcium phosphate sponges. Journal of Periodontology 71:410-417. Madihally, S. V., and H. W. T. Matthew. 1999. Porous chitosan scaffolds for tissue engineering. Biomaterials 20:1133-1142. Mao, H., K. Roy, V. L. Troung-Le, K. A. Janes, K. Y. Lin, Y. Wang, J. T. August, and K. W. Leong. 2001. Chitosan-DNA nanoparticles as gene carriers: Synthesis, characterization and transfection efficiency. Journal of Controlled Release 70:399-421. Norman, M. E., H. M. Elgendy, E. C. Shors, S. F. El-Amin, and C. T. Laurencin. 1994. An in-vitro evaluation of coralline porous hydroxyapatite as a scaffold for osteoblast growth. Clinical Materials 17:85-91.
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