Nanomedicine, Volume IIA: Biocompatibility

© 2003 Robert A. Freitas Jr. All Rights Reserved.

Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003 Implant Infection and Biofilms

In the late 20th century, millions of patients who received tissue and organ replacement worldwide experienced biomaterial-associated infection as one of the most destructive complications. Infections occurred in <1% of total hip replacements but in 2-4% of total knee replacements and in 7% of total elbow replacements [306], while Pseudomonas aeruginosa was the most frequent cause of bacterial keratitis in extended-wear contact lenses [309]. Vascular grafts became infected in 6% of specific risk groups, and intravascular catheters almost always became infected if not changed at regular intervals [306]. Ventricular assist devices developed infections 20% of the time in use under 31 days [307], and the Total Artificial Heart (TAH) of the late 1980s was at risk for infection 100% of the time if left in place for more than 1 month [308]. The formation of a biomaterial-associated biofilm (durable infection) usually led to removal or revision of the affected device or implant [238], with obvious devastating results for the patient.

Biofilms consist of bacteria embedded in a film of adhesive polymer, especially on implanted devices; bacteria within the film are protected from the action of antibiotics. As is well known in the industrial world, bacterial biofilms routinely foul many surfaces including ship hulls, submerged oil platforms, and the interiors of pipeworks and cooling towers, causing corrosion and metal component failure. Biofilms may infest gas biofilters [323], plastic mineral water bottles [324] and food processing systems [325]. But biofilm formation is also a serious medical problem that manifests itself as biomaterial-associated infections of devices (e.g., endotracheal tubes, sutures, intravenous catheters, urinary catheters, IUDs, and contact lenses), and as infections of prosthetic implants (e.g., mechanical heart valves, arteriovenous shunts and vascular grafts, joint replacements, biliary stents, dental implants, penile prostheses, and spinal implants) [313, 326-328], with Staphylococcus epidermis as the most common cause [310]. Depending on the organism involved, these infections can be either acute, with symptoms appearing relatively soon after material insertion, or chronic, with symptoms taking up to months to appear [238]. Electron microscopy of the surfaces of infected medical devices shows the presence of large numbers of slime-encased bacteria [331]. Biofilms may vary widely in thickness, limited more by nutrient transport than by surface roughness. For example, aerobic Pseudomonas aeruginosa biofilms can grow to 30-40 microns in depth as monocultures, but these biofilms can increase in depth to 130 microns when the culture is amended with anaerobic bacteria [2523].

Regular bacterial growth can sometimes be eradicated by cleaning implant surfaces with disinfectant or by systemic antibiotic administration. But bacteria may irreversibly adhere to artificial or natural surfaces that are surrounded by fluids. Unfortunately, adherent bacterial cells form biofilms preferentially on chemically inert surfaces [328], which possibly could include diamondoid materials useful in nanorobotics. For example, fungal biofilms are known to adhere to polystyrene plates [2553]. Once adhered, the bacteria can multiply, forming complex multilayered microcolonies and producing a slimy matrix material (usually a glycocalyx film) that encases the bacterial cells. This bacterial biofilm has been described as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface” [328]. Sessile biofilm communities are resistant to antibodies, phagocytes, and antibiotics [328] because the extracellular sulfated 20-kD acidic polysaccharide [332] slime matrix acts as a physical and chemical barrier to protect the bacteria from attack. Confocal optical sectioning shows that biofilms are highly hydrated open structures composed of 73-98% extracellular materials and void spaces [320]. AFM images of the surface structure of a hydrated biofilm [238] reveal numerous ~0.25-micron pores and ~0.50-micron channels. These discontinuous channels are believed to serve as nutrient-carrying passageways to all layers of the biofilm [319-323], thereby maintaining bacterial viability and capacity to proliferate. Atomic force microscopy has also been used to analyze the initial events in bacterial adhesion [333, 334]. Cells in different regions of a biofilm exhibit different patterns of gene expression [335] as well as functional heterogeneity.

All biomaterial surfaces, regardless of preparation, acquire patterns of organic and ionic contaminants whose distribution is directed by specificities of the outer atomic layers of the implant [306]. Glycoproteinaceous conditioning films – derived from fluid or matrix phases containing plasma protein such as fibrinogen, fibronectin, collagen, and other proteins – immediately coat a biomaterial or tissue implant [315] and act as receptor sites for bacterial or tissue adhesion [310].* The role of each constituent of this coating differs for each bacteria or tissue cell type. For instance, Staphylococcus aureus has discrete binding sites for collagen and fibronectin [311, 312]. Predicts Gristina et al [306]: “Modifications to biomaterial surfaces at an atomic level will allow the programming of cell-to-substratum surface events.” (See Section 15.2.2.)

* Proteins generally stick well to glass, less well to Teflon (Section, and least of all to mica.

Nanorobotic material surface characteristics and properties including roughness and surface area, fractal dimensionality, compactness or porosity, hydrophobicity, and chemistry may play a significant role in host cellular adhesion and in the ability of bacteria or cells to colonize nanorobotic surfaces [238, 316-318, 2587, 2588]. When tissue cells colonize a metal or polymer surface and integrate with the implant surface – whether via direct chemical interaction or host-derived macromolecules – then late-arriving bacterial cells are confronted by a living integrated tissue surface which resists bacterial colonization due to its viability, intact cell membranes, exopolysaccharides and functioning host defense mechanisms, and decreased availability of binding sites due to occupation of those sites by tissue cells. However, if bacterial adhesion occurs first and a stabilized microcolony has developed, late-arriving tissue cells cannot easily displace the primary colonizers to occupy and integrate the surface [306].

Once established, biofilm infections are rarely resolved by host defense mechanisms, even in strong hosts [331]. Antibiotic therapy typically reverses the symptoms caused by planktonic (individual) cells released from the biofilm, but fails to kill the biofilm itself [328-330, 1115-1117]. It is variously estimated that bacteria within biofilms are effectively from 20-1000 times [326] to 500-5000 times [1115] less sensitive to antibiotics than planktonic microorganisms. This reduced sensitivity appears to depend on physiological changes associated with slow growth in biofilm populations [1118-1120], possibly including gene derepression [5488] effects triggered by bacterial adhesion [1121]. Antibiotic-impregnated surfaces have enjoyed only limited success in resisting biofilm formation [2497-2512, 5291], in part because the supply of impregnating agent is nonrenewable. Nanorobotic devices capable of onboard resupply need not suffer this limitation.

One very successful surface treatment to combat the biofilm problem is a thin (~1-micron) ion-beam assisted deposition silver coating on PVC or polyethylene [5792]. Silver (Section and its compounds [336] have long been recognized as bactericidal. Silver-coated samples of implant material, tested in a modified Robbins device with Staphylococcus epidermidis, exhibited less prolific biofilm formation than did uncoated materials [238]. In 1997, Spire Corp. in collaboration with St. Jude Medical developed an ion-beam texturization process called “Silzone” [337] (originally “SPI-ARGENT” [314]) that allows the impregnation of heart valve sewing cuffs with silver metal to help prevent bacterial growth on the cuff. This reduces the incidence of postreplacement endocarditis, a life-threatening inflammation of the heart’s inner lining. Spire Corp. similarly treats central venous catheters and surgical guide wires to reduce the likelihood of clot formation and to increase lubricity, thus easing the insertion process [314]. The Erlanger silver catheter [338] and other silver-impregnated catheters [339-342] have demonstrated greatly reduced bacterial adherence and biofilm formation. In vitro tests of silver-coated polyurethane biliary stent material reduce adherent bacteria by 10- to 100-fold [343], and silver-coated Gore-Tex helps inhibit biofilm growth [344].

Permanently hydrophobized glass and ceramic surfaces have been found to largely prevent biofilm formation in the oral cavity environment [345]. Diamond is also very hydrophobic, though tests of biofilm formation on diamond have not yet been reported in the literature. A coating of ciprofloxacin-containing liposomes sequestered in polyethylene glycol (PEG) hydrogel that completely inhibits bacterial adhesion on silicone catheters has been demonstrated [346]. Alternatively, a correlation has been found between the enthalpy of adhesion (Sections 9.2.1 and 9.2.3) of bacteria to material surfaces and the strength of adherence of biofilm bacteria to those material surfaces [347]. In particular, there seems to exist a certain minimum bacterial adhesive tendency that is independent of the nature of the polymer surface; modified polymers with negative surface charge give a bacterial adherence close to the adherence minimum [347].

Another interesting approach from the nanomedical perspective is the application of low-power ultrasound in concert with aminoglycoside antibiotics (e.g., gentamicin) to enhance the effectiveness of antibiotic treatments and reduce the viability of sessile bacteria (e.g., Pseudomonas aeruginosa) by several orders of magnitude [1122-1131], a synergy known as the bioacoustic effect [1125, 1130]. In one experiment using 12 µg/cm3 gentamicin on biofilms, a 2-hour exposure to ultrasound at 70 KHz killed ~99% of P. aeruginosa biofilm bacteria at 100 W/m2 peak intensity, and ~90% at 10 W/m2 peak intensity, as compared to controls (e.g., without ultrasound) [1130]. These acoustic intensities are well below the <1000 W/m2 limit deemed safe in typical in vivo nanomedical power-supply (Section 6.4.1) and communications (Section applications. Anti-biofilm effectiveness declines log-linearly with increasing frequency: ~0.1 MHz acoustic waves are ~10 times more microbicidal than ~10 MHz waves, in combination with the gentamicin [1130]. It is postulated that ultrasound increases gentamicin transport through cell membranes (e.g., by high pressure, high shear stress, or cavitation), the proposed rate-determining step in microbial killing by the antibiotic [1130]. Electrical [1132-1139] and magnetic [1140] enhancement of antibiotic activity has been investigated. Pure sonication at 40 KHz also removes biofilms from food processing equipment [5625].


Last updated on 30 April 2004