Bioinspired Implant MaterialsBefuddle BacteriaResearchers are designing materials that either shed microbes orprevent them from adhering to device surfaces

James D. Bryers and Buddy D. Ratner

The use of biomaterials for implantsor medical devices stretches back35,000 years, when sutures wereused to close large wounds. TheGreeks and Chinese used gold in

dental restorations 2,000 years ago. However,since World War II, all manner of materials havefound use in implants or other medical devices,including ceramics such as alumina and hy-droxyapatitite; various metals such as stainlesssteel, titanium alloys, and cobalt-chromium-molybdenum alloys; and both syn-thetic and natural polymers suchas silicone rubber, Teflon, polyure-thane, and collagen. However,none of these materials was de-signed specifically as a biomaterialbut, instead, all were produced ini-tially for other consumer and com-mercial applications.

Engineered materials used inbiomedical implants or devicesmay inadvertently promote bacte-rial adhesion and biofilms. Anothercomplicating response to implanted medical de-vices comes from the body’s inflammatory system.Many such materials randomly adsorb host pro-teins and thus may “confuse” cell receptors onmobilized inflammatory cells. Hence, such im-plants typically trigger inflammatory responses,attracting neutrophils and macrophages thatattempt to engulf the implant. After macro-phage cells enter a stage of “frustrated phago-cytosis,” they eventually form multinucleate,foreign-body giant cells that ultimately be-come a tough, collagenous capsule that inhib-

its new vasculature and interferes with woundhealing.

Bioengineered Biomaterials

The realization that specific cell membrane re-ceptor-protein binding pairs dictate specific cel-lular responses has led researchers to designsynthetic biomaterials that mimic certain fea-tures of those cellular responses. To present oneor more specific signals to incoming cells, a

biomaterial must first resist ran-dom protein adsorption.

One way such bioinspired mate-rials seek to modulate host re-sponses is by presenting knownligands for cell membrane recep-tors, thereby affecting “outside-in” signal transduction pathwaysthat control cellular functions suchas adhesion, proliferation, andchemical release. Such materialsthus are also called engineered bio-materials because responses to

them are controlled with engineering precision.Consequently, next-generation biomaterials

will elicit specific cellular functions and directcell-cell interactions. Bioinspired approaches arebased on materials endowed with bioadhesivereceptor-binding peptides and mono- and oligo-saccharides. These materials can be patterned intwo and three dimensions to generate modelmulticellular tissue architectures, which may beuseful in generating complex organizations ofmultiple cell types.

Tissue engineering envisions the developmentof a new generation of materials or devices ca-

Next-generationbiomaterials

will elicitspecific cellularfunctions anddirect cell-cellinteractions

James D. Bryers isProfessor in theDepartment of Bio-engineering, theUniversity of Wash-ington and facultymember of the Uni-versity of Washing-ton Engineered Bio-materials (UWEB)Center, and BuddyD. Ratner is theWashington Re-search FoundationEndowed Professorof Bioengineering,Department of Bio-engineering andDirector, UW Engi-neered Biomaterials(UWEB) Center,Seattle, Wash.

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pable of the reconstruction of or integrationwith biological tissues. These devices will com-bine novel biomaterials (often biodegradable)with living cells to yield functional tissue equiv-alents. Such systems will be useful for organ ortissue replacement where there is a limited avail-ability of donor organs or where no naturalreplacements are available. Such constructs arealso useful as delivery vehicles for cytokines andgene therapy. Engineered biomaterials will beemployed in a variety of applications, includingnerve regeneration, artificial skin, orthopedicimplants, artificial pancreas and liver, gene ther-apy, vascular grafts, cornea replacement, inhibi-tion of coronary artery restenosis, neovascular-ization (by direct release of angiogenic factors ordelivery of specific genes expressing those fac-tors), and inflammation control.

Biomedical Device-Based Infections

Worldwide production of biomedical devicesand tissue engineering-related materials is anexpanding $170-billion-per-year industry, withmore than 5 million prosthetics implanted eachyear in the United States alone. However, 80%of hospital-acquired infections are associatedwith implants or indwelling medical devices,

with a fatality rate as high as 60%. Bacterialinfections are very common on implants, includ-ing prosthetic heart valves, orthopedic implants,intravascular catheters, left ventricular assist de-vices, cardiac pacemakers, vascular prostheses, ce-rebrospinal fluid shunts, urinary catheters, ocularprostheses and contact lenses, and intrauterinecontraceptive devices.

Until nonfouling biomaterials are availableand widely used, devices made with currentbiomaterials will continue to be subject to pro-tein fouling. The body typically reacts to pros-thetic implants by coating them with a filmconsisting of proteins such as fibronectin, vitro-nectin, fibrinogen, albumin, and immunoglobu-lins, many of which serve as binding ligands toreceptors on colonizing bacteria (Fig. 1, Step 1).Bacteria, transported to the substratum (Fig. 1,Step 2), adhere by either a nonspecific or specificbinding reaction (Step 3). If the bond is weak,bacteria may desorb into the liquid (Step 4).

Once attached firmly to the substratum, how-ever, bacteria begin cell-to-cell signal communi-cations (Step 5) that may control growth, repli-cation, plasmid conjugation, secretion ofvarious virulence factors, and secretion of extra-cellular mucopolysaccharides, which can form a

F I G U R E 1

Several distinct processes govern biofilm formation (see text ).

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tenacious three-dimensional gelatinous matrix(Figure 1, Steps 6, 7, and 8). These bacterialexopolymers can mix with those of other spe-cies, products of host cells, or blood platelets toform a mixed-cell-line biofilm that is highly re-sistant to rigorous antibiotic challenges. Ulti-mately, large sections of biofilm can detach (Step9) and be re-entrained in the bulk fluid, poten-tially contaminating other surfaces downstreamor, more insidiously, causing a thromboembo-lism and death.

Unfortunately, the advent of bioinspired ma-terials may inadvertently enhance bacterial ad-hesion and biofilm formation. Many of the mol-ecules (e.g., fibronectin, fibrinogen, vascularendothelial growth factor VEGF, and adhesionpeptide sequences such as RGDS and EILDV)selected to attract macrophage or fibroblastsand promote cellular growth may also enhancebacterial adhesion and colonization. Novelclasses of bioresponsive materials are now beinggenerated—at great research and developmentcosts—that may ultimately enhance microbialcolonization and infection. Such prospects war-rant equally innovative efforts to prevent micro-bial biofilm formation (see ASM News, March2004, p. 127).

Prospects for Controlling Bacterial

Adhesion and Preventing Biofilms

These emerging biomaterials require novel strat-egies to prevent biofilms. One 25-year-old ap-proach for preventing bacteria from colonizingsurfaces is to incorporate antimicrobial agents,either disinfectants or antibiotics, into the struc-ture or onto the surface of the biomaterial. How-ever, simply killing bacterial cells when they landon a device may promote antibiotic-resistant bac-terial strains and could lead to dead microbial cellsand their debris that accumulate on device sur-faces, thus promoting inflammation.

Thus, nonlethal ways of preventing microbialcell colonization are needed. Prospects include(i) surface material coatings that prevent adhe-sion, (ii) responsive surfaces that phase changeupon command, (iii) controlled orientation ofsurface-tethered adhesion molecules, (iv) negat-ing cell-cell quorum communication, and (v)interfering with receptor-ligand specific adhe-sion.

Stealth Materials Prevent

Bacterial Adhesion

When applied to surfaces or incorporated di-rectly within the biomaterial, poly(ethylene ox-ide) (PEO) can inhibit protein adsorption andbacterial adhesion. This polymer and its deriva-tives can be very effective, reducing adhesionfrom 60–90% for a variety of bacterial species.

For example, we have developed PEO-likeoligoglyme coatings [CH3-O-(CH2-CH2-O)n-CH3, in which n � 1–4], using radio frequencygas plasma discharge techniques that can applythese coating to a variety of base materials. Thedegree to which this family of coatings protectssurfaces depends in part on which oligomer isused and what bacterial species is being tested.In our studies with Pseudomonas aeruginosa, atetraglyme (n � 4) coating can reduce coloniza-tion by 99% relative to uncoated controls. Inother experiments, tetraglyme was deposited onglass in a pattern using photolithographic meth-ods before exposure to a suspension of Methyl-obacterium extorquens. After 72 hours, almostall bacteria were adhering to the bare glass, withvery few bacteria on the glyme-coated regions.

The elegance of the RF plasma technique isthat such PEO-like coatings can be depositedonto a variety of existing materials and geomet-ric shapes (e.g., inside and out of catheters,porous scaffold structures, metals, polymers,and ceramics). Although such glyme-plasmacoatings resist protein adsorption for at leastone week, longer-term bacterial challenges areneeded to more fully evaluate these coatings.

Specially Responsive Surfaces Change

Phase on Command

The surface-grafted, thermally responsive poly-mer poly(N-isopropyl acrylamide) (PNIPAM)has a critical solubility temperature of about32°C, making it insoluble in water at tempera-tures above 32°C but soluble at temperaturesbelow 32°C.

In lab tests, 90% of several microorganisms(Staphylococcus epidermidis, Halomonas ma-rina) and naturally occurring marine microor-ganisms that attached to PNIPAM grafted topolystyrene surfaces were removed immediatelywhen the hydration state of the polymer waschanged by simply rinsing the samples in cold(4 oC) water. Confluent sheets of mammalian cells

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can also be rapidly detached from sim-ilar PNIPAM-grafted surfaces whenthe temperature is shifted from 37°Cto room temperature. Whether tem-perature changes can remove thickbacterial biofilms from such surfacesremains to be tested.

Controlled Orientation of

Surface-Tethered Adhesion

Molecules

The goal in designing biomaterials isto enable their surfaces to interact indefined ways with incoming mam-malian cells and to eliminate randomadsorption of ambient proteins. Thiscommunication involves displayingspecific peptides or proteins on bio-material surfaces that will instigate adesired cell response.

For instance, one function of theprotein fibronectin (FN) is to adhereto a variety of molecules, matrices,and surfaces. Hence, FN and similarproteins (or their fragments) are be-ing tested in vitro for their ability toenhance mammalian cell adhesion,with little concern for whether theyalso enhance bacterial adhesion.Rather than unilaterally discontinuetheir use, perhaps these molecules may be mod-ified or specifically oriented in such a way as toenhance binding of mammalian cells while de-creasing that of bacteria. Fortuitously, FN bindsto mammalian cell receptors at epitopes on theFN molecule that are far removed from thatportion of FN that binds to S. epidermidis and S.aureus (i.e., the 29-kDa amino-terminus end).

If FN were bound to a biomaterial exclusivelythrough its amino terminus, this directed orien-tation might prevent adherence of S. epidermidisor S. aureus without interfering with mamma-lian cell adhesion. By coupling to silicon surfacesFab fragments of monoclonal antibodies thatwere generated against either the amino or car-boxyl terminus of FN, we created substrata thattethered FN molecules in a controlled orienta-tion—with all FN molecules exposing eithertheir amino or carboxyl termini to incoming S.epidermidis cells (Fig. 2). When FN is bound toa surface by its amino terminal, S. epidermidis

does not adhere. However, when FN is boundby its carboxyl terminus, S. epidermidis cellsadhere more avidly than to controls. Mean-while, mammalian fibroblast cells adhere andspread equally on both versions of the FN-treated surfaces.

Negating Bacterial Cell-Cell Quorum

Communications

Some bacterial populations can sense their size,density, and proximity, and also use signal mol-ecules, called pheromones or autoinducers, tocommunicate among members. This process,called quorum sensing, is required for biofilmsto form and for other virulence phenotypes to beexpressed in both gram-negative and gram-pos-itive bacterial species. Awareness of this processis leading investigators to search for agents thatmight interrupt these signals.

Although several signal molecule familieshave been identified in gram-negative bacteria,

F I G U R E 2

Staphylococcus epidermidis adhesion in a flow cell device (shear stress 2 N/m2) to glasssurfaces presenting oriented fibronectin (FN) molecules. FN-NH3 � silane-treated glasssurface with FN bound by available amino groups (random orientation). FN-COOH �silane-treated glass with FN bound by available -COOH groups (random orientation).FN-FabCOOH � glass surfaces with FN held by its -COOH terminus (controlled orientation).FN-FabNH3 � glass surfaces with FN held by its NH3 terminus (controlled orientation).

Volume 70, Number 5, 2004 / ASM News Y 235

the most intensively studied and best under-stood are those that belong to the N-acylhomo-serine lactone (AHL) family. AHLs contain ahomoserine lactone ring attached via an amidebond to an acyl side chain containing from 4 to14 carbons. Some natural products antagonizeAHL-mediated quorum sensing. For example,halogenated furanones that are structurally sim-ilar to AHLs and are produced by the macroalgaDelisea pulchra can inhibit AHL-regulated pro-cesses, including biofilm formation. Althoughresearchers have produced many synthetic fura-none compounds, some of which partly blockbiofilm formation, many of these compoundsalso provoke inflammatory responses frommammalian cell lines.

Unlike gram-negative bacteria, gram-positivebacteria use peptides rather than AHLs as signal

molecules to regulate physiological pro-cesses, such as bacterial competence,sporulation, initiation of plasmid conju-gation, and virulence gene regulation. Forinstance, under control of quorum sens-ing, both S. aureus and S. epidermidisproduce virulence-related cell wall deriv-atives and exoproteins.

This process, controlled through theaccessory gene regulatory (agr) operon,also controls biofilm formation in S.epidermidis. Meanwhile, S. aureus usestwo quorum-sensing mechanisms—thefirst consists of the autoinducer RNAIII-activating peptide (RAP) and its targetprotein, while the second consists of thepeptide AIP and its receptor, AgrC, undercontrol of the arg operon. The heptapep-tide RNA III inhibiting peptide (RIP) caninhibit virulence and biofilm formation inS. aureus and S. epidermidis. RIP com-petes with RAP, inhibiting phosphoryla-tion of an intermediate protein and lead-ing to reduced adhesion and inhibition ofRNA III synthesis, which leads to thesuppression of toxin synthesis.

Tests of synthetic analogs of thesesignal peptides have proved frustratingbecause, while one analog may be suc-cessful in down-regulating virulencefactor expression, it typically will up-regulate biofilm formation. Such ana-logs also seem to have contradictoryeffects when applied to different bacte-rial strains or species. While no ideal,

noninflammatory analog inhibitor of gram-pos-itive or gram-negative bacterial quorum signal-ing has yet been identified, the potential impactof such agents has fostered intensive research byboth academic and private industry. If suchagents could be identified, they might be incor-porated into implanted devices and slowly re-leased to prevent biofilms from forming.

Strategies for Interfering with

Receptor-Ligand Specific Adhesion

The alarming increase in antibiotic-resistantbacteria has stimulated the search for novelways to prevent infections. One attractive strat-egy calls for developing agents that interferewith the ability of pathogenic bacteria to adhereto host tissues, blocking this early and crucial

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stage of the infection process. Numerous exper-iments, including those that use ligand and ad-hesin analogs, anti-adhesion antibodies, andadhesin-based vaccines, validate this approach.Although such research is progressing, similarefforts aimed at preventing microbial pathogensfrom adhering to biomedical devices is lagging.

Bacterial and other microbial pathogens useadhesin receptors to bind specifically to moleculesthat adsorb to medical devices. For example, whenP. aeruginosa cells bind to a particular glycolipidalong the cornea, they can give rise to infectionsfor those who wear contact lenses. Similarly, S.epidermidis or S. aureus cells can bind specificallyto various blood plasma proteins (e.g., fibronectin)that adsorb to cardiovascular devices.

To determine whether such specific bindingcan be prevented, we generated a series of mono-clonal antibodies (MAbs) to the FN-binding re-ceptor of S. epidermidis. These MAbs reduced S.epidermidis biofilm formation by only about 50%(Fig. 3). To improve inhibition, we have taken thespecific antigen-binding sites from different MAbs(that bind to the antigen at different epitopes)and formed smaller, single-chained, bivalentantibody fragments (“diabodies”). Certaindiabodies inhibit biofilm formation by 99%.We envision developing a biomaterial capable

of steadily releasing such adhesion-blockingmolecules as a way of continually protectingbiomedical devices.

Such strategies that aim at blocking specifictypes of adhesion offer an attractive means ofpreventing pathogens from colonizing biomedi-cal devices. If effective, this strategy could helpto reduce the use of antibiotics and therebylower the likelihood that antibiotic-resistantstrains would emerge among individuals whosetreatments require the use of indwelling devices.The major drawback of this approach is thatmany colonizing microorganisms possess morethan one type of adhesin-ligand combination,which might require developing and using mul-ticomponent, anti-adhesion cocktails.

Tissue engineering and the generation of en-gineered biomaterials may inadvertently gener-ate biomedical implants or devices that presentinterfaces that promote bacterial adhesion andbiofilm formation. New trends in attractinghealing mammalian cells must be judiciouslyconsidered for their potential to promote bacterialcolonization. Emerging technologies directed atnegating one or more processes in biofilm forma-tion may prove successful without resorting to theapplication of toxic agents or antibiotics.

SUGGESTED READING

Hendricks, S. K., C. S. Kwok, M. Shen, T. A. Horbett, B. D. Ratner, and J. D. Bryers. 2000. Plasma-deposited membranes forcontrolled release of antibiotic to prevent bacterial adhesion and biofilm formation. J. Biomed. Materials Res. 50:160–170.Johnston, E. E. 1997. Surface and biological properties of biofouling-resistant, PEO-like plasma deposited films. Ph.D.dissertation, University of Washington, Seattle.Kwok, C. S., C. Wan, S. K. Hendricks, J. D. Bryers, T. A. Horbett, and B. D. Ratner. 1999. Design of infection-resistantantibiotic-releasing polymers: I. Fabrication and formulation. J. Controlled Release 62:289–299.

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