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BIOMATERIAL SURFACES AND INFECTION

The purpose of this term paper is to give an overview of the mechanisms involved in biomaterial-centered infection (BCI) and review the approaches to solve the problem via development of antimicrobial surfaces and post-implantation procedures. It should be noted that the paper is not about sterilization or other such pre-implantation procedures related with prevention of bacterial infection.

Infection is one of the major problems in the biomaterial science. It is still the most common cause of biomaterial implant failure in modern medicine. For instance, about 1 in 5-10 urinary tract catheter implants results in an infection. To give another example; the performance of one of common implants, hip joint replacement depends on four major factors: fracture, wear, loosening of the implant and infection.

The incidence of infection depends in which body part the material is implanted. In other words, the probability of those microorganisms to reach the biomaterials surface. The colonization of medical implants can be hardly avoided when parts of these implants are exposed to the nonsterile exterior such as urinary catheters. Bacteria attach to the exterior parts of the implants, migrate along the surfaces into the interior parts of the body and cause infection. On the other hand microorganisms cannot easily reach hip prostheses. Thus the incidence of BCI varies from 4% for hip prostheses to 100% for urinary tract catheters after 3 weeks use.

Table 1. Incidences of infection of different biomedical implants and devices. Adapted from Dankert et al.

* From experiments in calves and sheep.

Microbes colonize on the suitable surface of the implant and may cause severe problems. The complications caused by BCI may vary from disfunctioning of the implanted device itself to lethal sepsis of the patient. This phenomenon of infection by microbes accumulated on biomaterial is commonly named as biomaterial-centered infection (BCI). Treatment of BCI is more complicated than infection of planktonic microbes as microorganisms adhered to the surface are more resistant to antibiotics. Consequently, it seems that the most suitable solution to the problem is the removal of the infected implant at the expense of considerable costs and patient’s suffer if it is possible at all, treatment of it in vitro and re-implantation or exchanging it with new one. Increased health budgets also result as hospital care and drug costs increase. However, a more convenient way to deal with the BCI problem is to prevent attachment and colonization of microorganisms at the initial steps. Scientists and manufacturers, thus, try to develop biomaterial surfaces onto which bacteria cannot attach themselves, grow and colonize easily if not to prevent the attachment altogether.

Combat Approaches ^[9]

There are different approaches in combating biomaterial-centered infection. Here we will examine most prominent ones and compare them.

Antimicrobial Chemical Release

Continuous release of antimicrobial agents from biomaterial is one approach to prevent microbial infection on the material. This antimicrobial agent is mostly antibiotics. Antiseptics are also used but are restricted since they are not selective in their toxicity and might damage host cells also. The problem with this approach is that after some time ‘sub-inhibitory’ concentrations of antimicrobial agents released into to the surrounding tissue or fluids may lead to development of resistance strains of microbes. This approach is especially pronounced in implants which are intended to be used for longer time periods. Partial solution to the problem is to use more than one antibiotic compound thus broadening spectrum and reduce chance of resistance build-up. In fact it is a common practice in medical applications, treatments to prevent microbial resistance formation.

Immersion

The clinical success of this approach has been varied. Although immersion is one of the most straightforward methods for loading antimicrobial agents into medical devices, biomaterials generally have limited affinity for these agents, and the majority of the drugs will be adsorbed on the surface and not diffused into depths of the polymer matrix. Antibiotics such as rifampicin, ciprofloxacin, tobramycin and certain cephalosporins have been examined. In general, studies have illustrated that the immersion of polymers in antimicrobial solutions, although reducing early-onset colonization of devices, would be unlikely to prevent biofilm formation in long-term implants. For an optimum effect, loading of medical devices by immersion in aqueous antibiotic solutions should be restricted to medical devices that are hydrophilic in nature, i.e. possess a hydrophilic (hydrogel) coating allowing absorption of the aqueous solution. Immersion of hydrophobic medical devices in aqueous antibiotic solutions will result in a weak and limited surface attachment of antibiotic to the device with poor clinical effects. However, the method is very effective in preventing bacterial intrusions immediately prior to the implantation of biomaterial.

Surface Coatings

Specific coatings may be applied onto a material to receive antimicrobial agents to provide device protection from infection. Drug loading is enhanced by pre-coating the surface with a tie layer, wherein the interaction between the antimicrobial agent and the tie layer is facilitated by electrostatic interactions. Anionic antibiotics can be bonded electrostatically to the surface of the medical device via a tie layer composed of cationic surfactants such as tridodecylmethylammonium chloride (TDMAC). TDMAC can also bind antibiotics if added after device placement by local irrigation or by systemic administration. Several studies have examined the clinical efficacy of catheters coated with antimicrobial agents using this coating technology. For example, polyurethane catheters coated with minocycline and ethylenediaminetetraacetate showed potential in reducing recurrent vascular catheter-related bacteremia. Minocycline and rifampicin coatings have been shown to significantly reduce the risk of catheter-related infections of the bloodstream and maintain effective antimicrobial activity against colonization of indwelling central catheters for at least two weeks. Minocycline coated onto urethral catheters has been shown to provide some protection against colonisation6, and Johnson et al. described substantial in vitro antimicrobial activity of a commercially available nitrofurazonecoated silicone catheter against problematical multidrug-resistant clinical bacterial isolates.

A number of other antimicrobial agents have been included in coatings of medical devices. The antibacterial activity of silver-containing compounds as antimicrobial coatings for medical devices have been widely investigated but with conflicting results, especially in vivo. Silver suphadiazine used in combination with chlorhexidine has received particular interest as a central venous catheter coating mainly due to the broad spectrum of combined activity against organisms that cause catheter infections. Some conflicting results have appeared in studies with this combination, and a recent interesting study showed the incidence of catheter-related bloodstream infection associated with minocycline/rifampicin catheters was significantly lower than for their silver sulphadiazine/chlorhexidine-coated comparators. This difference may, in part, be due to the nature of the coating on these catheters. In minocycline/rifampicin catheters, both the external and internal surfaces were coated, whereas the silver sulphadiazine/chlorhexidine catheters were coated only on the external surface, illustrating the importance of the nature of the catheter coating on the resultant clinical efficacy of such systems. Nevertheless, antibiotic coating of devices is still a major focus of attention in antimicrobial research to combat BCI.

Matrix Loading

The loading of antimicrobial agents into biomaterial by immersion or coating technologies has the advantage of being relatively simple. However, the limited mass of drug that can be incorporated may be insufficient for a prolonged antimicrobial effect, and the release of the drug following clinical insertion of the device is rapid and relatively uncontrolled. A means of reducing these problems is by direct incorporation of the antimicrobial agent into the polymeric matrix of the medical device at the polymer synthesis stage and/or at the manufacture stage.

A principal disadvantage to be considered of the direct incorporation of antimicrobial agents into the matrix is the reduction in mechanical properties of the host polymer. The mechanical properties of the polymer are essential to ensure the optimal performance of the medical device so the potential hazards of incorporation of antimicrobial (and related) agents must be considered.

            Drug Polymer Conjugates

Permanent coatings incorporated into the structure of material surface covalently serve good antimicrobial purposes and is long lasting. Covalent attachment of drug to the surface of polymer has received comparatively little attention in contrast to the other methods of drug incorporation into biomaterial structure. The covalent linkage of an agent to a monomer prior to polymerization provides a method of producing perhaps the most resilient drug-polymer material. Coronary stents have been modified to have antithrombogenic and antibacterial activity by covalent attachment of heparin to silicone with subsequent entrapment of antibiotics in cross-linked collagen bound to the heparinized surface.

            Drug-polymer conjugates have been found to provide significant reductions in bacterial adherence and urinary encrustation, which offers potential for urinary catheter use. There are certain limitations to this approach, such as the selection of therapeutic agents with chemistry that is compatible with the synthetic reaction scheme, and there is a greater expense associated with the synthetic process. We will examine some promising studies concerning this approach.

Functionalization by Particle Bombardment

            Gamma radiation and glow discharge techniques act by introducing new functional groups onto the polymer surface, and the newly created functional groups may possess intrinsic antimicrobial activity. Antimicrobial substances may also be linked covalently to the functional surface groups to provide increased antimicrobial activity. A photochemical approach to coatings has been demonstrated to cause significant reductions in bacterial attachment to polymers and this could bring further exciting benefits. However, to ensure patient safety, it is recommended that anti-adherent coatings should be combined with site-specific delivery of antimicrobial agents.

            Biomimetic biomaterials

            Another novel and interesting approach in combating BCI is the use of “biomimetic” materials as itself or as coatings. Biomimetic materials are the ones that mimic the characteristics of natural tissue. Such materials would be extremely biocompatible if properly manufactured. These coatings can also be combined with antimicrobial agents rendering them as good biocompatible infection-resistant material.

            An interesting example of this type of material is the novel silicone technology developed one. This relies on higher-molecular-weight polysilanes as cross-linking agents for silicones. These silicones imitate the body’s natural defense mechanisms by exuding to the surface a substance that is subsequently shed and replenished but thereby releasing attached bacteria and mimicking the natural shedding of tissue cells and mucus. The exudates can be extremely lubricious, exhibiting coefficient of friction values that are less than 0.1. This allows an optimal ease of device insertion and removal without tissue trauma, and antimicrobial agents and other drugs can be delivered to the biomedical device surface for site-specific activity.

Mechanism of the microbial adhesion ^[5]

Once microorganisms found a way to reach nearby of the biomaterial surface a multi-step process starts leading to the formation of a complex, adhering microbial community that is termed as ‘biofilm’. A biofilm can be defined as a layer of prokaryotic or eukaryotic cells, anchored to substratum surface and embedded in an organic matrix of biological origin. The organic matrix also termed as ‘conditioning film’ might originate from infecting bacteria or the host itself. These infecting bacteria produce extensive slime like organic material - exopolysaccharides often called ‘glycocalyses’, in combination with the derivatives of host environment. Thus they are able to form a confluent biofilm on tissue or medical devices. Glycocalyses             are important virulence factors of BCI.

In the medical and dental fields research is focused most often on how to prevent and control formation of an infectious, pathogenic biofilm, and on how to keep the commensal microflora of the skin, urinary and intestinal tracts, or oral cavity intact and free of potential pathogens. In the oral cavity, daily tooth brushing is required in order to remove dental plaque and prevent the formation of a cariogenic or periodontipathogenic biofilm. Some bacteria already adhered to the surface or sessile microorganisms may stimulate the adhesion of planktonic microorganisms present in saliva in vast amounts.

Although the function and appearance of biofilms in various environments may be different, all biofilms originate from the same sequence of events. When microorganisms and substratum surfaces are in an aqueous environment, in which organic matter present (e.g. tear fluid, urine, blood, or saliva), substratum surfaces will first become covered with a layer of adsorbed organic molecules, ‘conditioning film’. The adhesion of organic molecules take place before microorganisms adhere simply because transport and adsorption of molecules to a substratum proceed relatively fast compared to that of microorganisms. The next step in the formation of biofilm structure is the transport of microorganisms towards substratum. The forces involved in this transport can be various such as Brownian motion, gravitation, diffusion, convection or the intrinsic motility of a microorganism. Alternatively, also microorganisms in suspension may be transported towards each other and microbial (co)aggregates can be formed. Subsequently, microbial adhesion (either of single microorganisms or of (co)aggregates) may occur. The aggregates are initially reversible which becomes irreversible in time due to excretion of exopolymeric substances by the adhering microorganisms. Thus sometimes bacteria itself can form conditioning film as opposed to host or environmental film. When a conditioning film is present, an adhering microorganism is usually not in contact with the actual substratum surface and the strength of biofilm formation becomes dependent upon the cohesiveness of the conditioning film, rather than upon its direct interaction with the bare substratum surface. Eventually, adhering microorganisms start growing which is the major factor, contributing to the accumulation of a high number of cells on a substratum surface.

            Biofilm formation serves great advantages to the infecting bacteria. Rapidly dividing bacteria can more easily spread along the surface of the material within the biofilm. Some bacteria that are shed with glycocalyx and may become free gain a chance to adhere to the new uncoated, non-colonized surfaces. Biofilm serves an ion-exchange matrix within itself thus providing more organic nutrients and also enable bacteria to counter cationic antimicrobial agents. The immediate implication of the process is the prolonged and high levels of antibiotic treatment. That is they are now much more potent enemies. Bacteria within biofilms can be 1000 times more resistant to antibiotics than their planktonic counterparts. The biofilm structure also provides close proximity of other microorganisms thus high cell density co-operative activities, such as cross-breeding and genetic exchange and resistance transfer.

Roughness has an influence on biofilm formation. However, it seems to be of minor importance with regard to initial adhesion. Micrographs of organisms adhering to substratum surfaces seldom show a preference of microorganisms to adhere in scratches of grooves. The influence of surface roughness on biofilm formation is likely more related to the difficulties involved in cleaning rough surfaces, resulting in rapid re-growth of a biofilm, rather than to being a contributing factor directly.

Biomaterial surface charge and infection ^[2]

The overall charge of bacteria results from relative amounts of anionic and cationic surface molecules of the cell surface. Usually these molecules are either acidic or basic structures. Consequently, the effective charge under the influence of specific adsorption of ions and strongly depends on pH and ionic strength of the medium.

Most bacteria carry a net negative surface charge at physiological temperature and hydrogen ion concentration. Therefore, negatively charged biomaterials on their surface discourage adhesion, while positively charged surfaces promote it. Current approaches to the development of new antimicrobial surfaces are based predominantly on developing non-adhesive surfaces. Adhesion, however, is only one of the first steps in the formation of a biofilm infection and in order for a biofilm to develop fully, the adhering bacteria should colonize. Surface growth of the initially adhering bacteria was found by Harkes et al. to be absent on positively charged poly(methacrylates) for Escherichia coli. Barton et al. found that surface growth of Pseudomonas aeruginosa correlated with the free energy of adhesion, while no such correlation was found for Staphylococcus epidermis and E.col . Gottenbos et al. [2] reported that growth of P.aeruginosa on biomaterial surfaces decreased with the increasing strength of adhesion to the surface. In this paper they demonstrate that in order to develop surfaces with a low risk of infection,  in vitro studies should not only take into account initial adhesion, but also loot at surface growth of the adhering microorganisms.

            In their study Gottenbos et al. have used four bacterial species S. aureus, S. epidermidis, E. coli and P. aeruginosa all of which were negatively charged in phosphate-buffered saline (PBS) medium and had -10 mV, -8 mV, -16 mV and -7 mV respectively. The biomaterials used were methyl methacrylate homopolymer (PMMA) and copolymers of methyl methacrylate (MMA) with either 15 mol% methacrylic acid (PMMA/MAA) or 15 mol% trimethylamino ethyl methacrylate chloride (TMAEMA-Cl).

The measurements of water contact angle show that all the polymers films are intermediately hydrophobic surfaces. And the hydrophobicity shows no major variation with ξ potential. This indicates that all results could be interpreted without the interfering influences of substratum hydrophobicity. X-ray photoelectron spectroscopy analyses indicated that the positive charge originated from nitrogen-containing groups, while the increased negative charge was caused by oxygen-containing groups on the modified acrylate surfaces. Using four different bacterial species and three polymer films with different charges but similar composition, the scientists screened adhesion and colonization processes during up to 7 hours.

Figure: P. aeruginosa on negatively charged PMMA/MAA (– left), PMMA (- center) and on positively charged PMMA/TMAEMA-Cl (+ right).The bar represents 10 µm.

            The first two hours can be regarded as the adhesion period. In this respect, we observe less bacterial adhesion on negatively charged polymers in the above images. On the other hand, more bacteria adhered to the positively charged PMMA/TMAEMA-Cl on the right. After 4 hours, however, only on the negatively charged polymers bacteria are able to colonize. The implications of this observation are also reflected in the graphs below.

Figure: Number of adhering bacteria on negatively charged PMMA/MAA (– –) and PMMA (–) and on positively charged PMMA/TMAEMA-Cl (+) in a parallel plate flow chamber. The dashed lines indicate the time period during which PBS was perfused through the flow chamber before the introduction of growth medium. (a) S. aureus ATCC 12600; (b) S. epidermidis HBH₂ 102; (c) E. coli O2K2; (d) P. aeruginosa AK1

All the graphs demonstrate the tendency of bacteria to adhere to the positively charged surfaces. Colonization, however, is preferred generally on negatively charged surfaces although their initial adhesion is impeded by the negative potential of these polymers. It is especially pronounced for (d) P. aeruginosa. It is also worth noting that bacteria tend to colonize on moderately negative polymers (PMMA) rather than on PMMA/MAA which have higher amount of negative charge on it. Complete inhibition of growth here, as found here for Gram negative bacilli P. aeruginosa, possibly indicates that elongation of adhering bacteria, necessary for cell division is prevented by strong binding through attractive electrostatic interactions.

Initial adhesion has always been recognized as an essential step in biofilm formation. The study, in the contrary, demonstrates that adhesion and surface growth may be oppositely affected by substratum charge. When a biomaterial surface is negatively charged it is likely that most bacteria won’t bind them as readily as to the positively charged surfaces. This may delay the formation of biofilm. Positively charged surface are more adhesive for bacterial cells. However they impede the colonization of Gram-negative bacilli by the strong electrostatic attractive forces. As biomaterials are often inoculated during implantation surgery where they don have yet conditioning film to mask initial surface, positively charged surfaces may play role in preventing bacterial proliferation. This may also important in cases where the absorbing proteins are not abundantly present such as urinary catheters and voice prostheses for laryngectomized patients with hampered salivary flow due to irradiation.

Functionalized biomaterials ^{[3, 4, 7, 10]}

Attaching some functional groups to the surface of the biomaterials is another approach in preventing BCI. These functional groups act as barriers for bacterial adhesion, colonization. For instance, polycations and especially quaternary ammonium compounds possess strong antimicrobial effects.

            In one study, Flemming and his/her colleagues [10] examined the effect of some functional groups attached on polyurethane polymer material. Polyurethane material is one of the leading compounds used in composition of medical devices. In addition, polyurethanes seem to perform fairly well against bacterial adhesion when compared to other polymers.

Quaternary ammonium groups are widely known as disinfectant compounds. Here, attached on polymer surface they evidently serve that function also at least in vitro. This approach of attaching functional groups like quaternary ammonium groups on biomaterials as a antimicrobial agents, have many advantages. There are no antimicrobial agents leaching from the surface, thus providing long-term protection against bacterial colonization. Furthermore, attachment reduces the risk of developing antimicrobial resistant strains, as the concentration of the antimicrobial groups is constantly above the minimal inhibitory concentration. Quaternary ammonium groups possess strong positive charges. This is proposed to inhibit the clinical isolates bacteria that are usually negatively charged by exerting strong adhesive forces on them and thus preventing division, especially in the case of rod-shaped bacteria.

Graph: Colonization of pellethane, sulfonated pellethane and 50% methyl quaternized polyurethane by S.aureus.

Pellethane and sulfonated pellethane are used as control groups in the experiment. Sulfonation is used because they possess strong biological effects and serve as negative controls. The efficiency of inhibition of bacterial proliferation of quaternized polyurethanes is clearly demonstrated in the above graph. These functional groups effectively prevent bacterial interaction at both steps: adhesion – the time after one hour, and colonization – after 24 hours in the graph.

Graph: Colonization of pellethane, phosphonated pellethane and zwitterionic phosphonated polyurethane by S.aureus.

After one hour of inoculation of bacteria we observe higher number of microorganisms on zwitterionic phosphonated polyurethane (GPC-PU) than on phosphonated pellethane and pellethane. However, the initial trends in bacterial adhesion do not necessarily imply subsequent similar trends in long-term colonization. This fact was also shown in the study of Gottenbos et al. (2001) which we have examined previously. After 24 hours there is a decrease in CFU number for GPC-PU as opposed to a sharp increase in the case of pellethane and phosphonated pellethane.

            The scientists also have carried out experiments on materials treated with albumin to mimic conditioning film as in vivo conditions. The results, however, were not promising. Albumin treatment of polyurethanes with ethyl quaternary ammonium led to a complete loss of antimicrobial activity. They masked the functional groups on polymers and thus preventing bactericidal activity of quaternary ammonium groups.

            A recent paper published in Biomaterials journal caught my attention. This 2002 paper of Gottenbos et al. [7] demonstrates antimicrobial activity of quaternary ammonium functional groups both in vivo and in vitro.

            In this paper, the antimicrobial activity of silicone rubber with a covalently coupled 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) coating was studied. In contrary to the experiments of Flemming et al., they studied basal polymer material with and without QAS coating both with human plasma protein and without them. Moreover, they tested them in rats as well. Furthermore, the adsorption of human proteins had no effects on the antimicrobial activity of the biomaterials.

            In order to test antimicrobial properties of QAS, silicone rubber polymer (also a widely used biomaterial) with or without QAS were seeded on with four bacterial species that are typical infectious bacterial agents of human: Staphylococcus aureus, Staphylococcus epidermidis, E.coli and Pseudomonas aeruginosa. These species are most widely reported bacterial species in infection incidences in human. The same experiments were carried out on human plasma protein treated samples of QAS coated silicone rubber. In vivo experiments were done on rats by subcutaneously introducing polymer implants and seeding pre- or postoperatively with S.aureus.

            As a result, it was clearly demonstrated that QAS coated polymer implants is promising in preventing biomaterial centered infection. It is worth notifying that QAS is a strong positively charged material which possibly acts as potent inhibitor through strong adhesion of bacterial cells.

                        In the table 1 we can clearly see that one of the most vulnerable implants to BCI is the urinary tract catheters. Consequently, researchers seek ways of decreasing the number of incidences. Multanen et al. [3] tried to prevent the phenomena by coating bioresorbable self-reinforced poly-L-lactic acid (SR-PLLA) polymer urological stents with silver nitrate. Some relatively temporary obstructions to the bacterial adhesion used are immersion of stents in a suitable antibiotic solution, salicyclic acid impregnation or electrification. These approaches are essentially helpful to prevent bacterial protrusion during surgical operation. However, as it is noted previously bacteria are able to attach to the exterior parts of the implants, migrate along the surfaces into the interior parts of the body and cause infection. Permanent surface coatings seem to be a solution to the problem.

            Bioresorbable polymers have been used as surgical suture materials since the 1960s. Extensive animal studies have shown good biocompatibility of polygycolides and polylactides. To give better mechanical strength to bioabsorbable material, several self-reinforcing (SR) techniques can be used. In an experimental study of Kemppainen et al. (1992), self-reinforced poly-L-lactide spiral stents (SR-PLLA) showed good tissue penetration and biocompatibility properties. SR-PLLA stents have been used clinically in the treatment of recurrent urethral strictures with optical uretromoty and combined with finasteride in the treatment of acute urinary retention.

Four bacterial strains isolated from patients with urinary tract infection were used. Silver nitrate was blended into ε-caprolactone/L-lactide copolymer and used as coating for SR-PLLA. The observations revealed that the coating significantly reduced the bacterial adhesion. The inhibition was enhanced with higher concentrations (10 and 5 weight %). However, one of the bacterial species used, namely Enterococcus faecalis was problematic. Even the highest concentration has no much effect on the bacterial growth.

             Silver nitrate coated SR-PLLA material also reduced bacterial cells in ambient artificial urine, interestingly. In conclusion, this finding may reduce amount of stent-centered urinary tract infections.

            Further study of Multanen et al. [4] on biocompatibility issues of silver nitrate coated SR-PLLA rods. They have also tested ofloxacine coated SR-PLLA rods. SR-PLLA rods coated with pure poly(caprolactone-co-L-lactide) or blended silver nitrate (10, 5 or 2 weight %) or ofloxacine (5 or 2 weight %) were implanted in the dorsal muscles of male rabbits.

            It is known that both silver nitrate and ofloxacine coatings reduce bacterial adherence to SR-PLLA. And the study revealed that blending silver nitrate or ofloxacine up to 5 weight % to caprolactone-L-lactide coating of SR-PLLA does not have any essential effect on the biocompatibility. Thus, coated SR-PLLA seems to be a good antimicrobial biomaterial candidate for urinary tract implants.

            Functionalization by Particle Bombardment ^[11]

            Polymers are the most widely used biomaterials due to their mechanical viscoelasticity properties that closely resemble natural tissue’s ones. As it is mentioned above silver is a good coating candidate for biomaterials since it possesses antimicrobial activity and low level toxicity to human tissues.

            Novel surface engineering techniques involving ionized particle bombardment are particularly interesting to induce new surface properties without altering bulk properties since only a small thickness of the material is involved. In these techniques an ion source is used to produce the appropriate ionized species, which can be separated with a mass separator, before being accelerated in an electrical field. The delivered ion beam induces modifications on the macromolecular structure through: gas evolution, formation of double bonds, chain scissions or cross-linking. A prominent feature of the technique is that a thickness corresponding to the penetration depth of the ions can be efficiently regulated. In polymers, high modifications of the mechanical behavior are initiated by limited molecular modifications, leading to properties degradation or improvement chain reactions are dominated by scissions or cross-linking.

            Davenas et al. used in their modeling study of modifications induced by ion bombardment or implantation of silver ions in poly(ethylene) - PE. PE has been chosen as a model because of its simple structure and absence of additives allowing clear identification of processes at the molecular level.

The scientists used a broad range of surface analysis techniques to characterize the modifications of the polymer alone or the action of silver implantation. The comparison of PE films irradiated at same energy and ion fluence with Kr⁺ and Ag⁺ allowed the separation of these two effects, since differences between these treatments could be attributed to the action of silver, as changes resulting from energy transfers are nearly the same for these ions.

Figure: Optical absorption spectra of PE films implanted at 15 keV with: (1) 5x10¹⁵ Ag⁺ cm^-2: (2) 2x10¹⁶ Ag⁺ cm^-2.

In the graph of optical absorption spectra we observe a strong increase in the absorption in the UV region extending to the visible region. This absorption can be attributed to the graphitic component formed. It is noted that as the irradiation is lowered to 10 keV, the analysis of the UV edge showed that this carbon layer became more diamond-like. This in turn lead to reduced bacterial adhesion.

The formation of metallic silver particles appearing at fluences larger than 10¹⁶ Ag⁺ cm^-2 is evidenced by different analytical techniques. Increased antibacterial effect resulting from colloidal silver has been detected. When the implantation energy was lowered, leading to increased accessibility to the surface colloidal silver, reduction of bacterial colonization has been observed.

Are all the clinical isolates of bacteria negatively charged? ^[1]

            Jucker et al. have tested adhesion of the Stenotorphomonas maltophilia to glass and Teflon. This bacterial species was isolated from a catheter specimen from a patient with a suspected urinary tract infection. Indeed, Stenotorphomonas genus is known to be involved in human infections. An interesting fact concerning the species is that they are positively charged at physiological pH. Taking into account the fact that pH and ionic strength in the medium where from the bacteria were isolated vary greatly; the researchers have investigated charge dependency on these factors. In adhesion experiments performed with glass and Teflon they showed that the positive charge of S. maltophilia has promoted its adhesion.

Figure: Contact angles θ_w plotted against the electrophoretic mobilities u of gram-negative (triangles) and gram-positive (circles) clinical isolates (open symbols) and environmental samples (filled symbols).

The charge on the cell surface of bacterium was measured as the mobility u of bacterium in an electric field. The above graph illustrates how different is the charge on the bacterial surfaces of both clinical and environmental isolates. This wide range of bacterial surface properties makes it difficult to design solid surfaces resistant to bacterial colonization. In addition, any surface will be coated by organic compounds (conditioning film) in the body by serum or tissue proteins and glycosaminoglycans. Thus the surface properties of the implant become unpredictably changed and complex and make the control of bacterial adhesion very difficult. The scientists advise here to accept the fact that colonization of solid surfaces cannot be completely prevented. Some solutions to the problem are: coating biomaterial with bactericidal chemicals where uncontrolled bacterial colonization is detrimental; development of materials with weak bonding between their surfaces and the biofilm to achieve detachment of the biofilm at low shear forces; coatings of easily detaching or dissolving organic layers which will prevent the development of undesired long-term stable biofilms.

Biomaterials topology and infection ^[6]

Surface topological properties also play role in promoting or preventing biomaterial centered infection. Highly porous materials are potential caves for bacteria into which white blood cells hardly can reach. On the other hand, such porous and/or rough materials serve less surface area for initial bacterial adhesion.

            Bellon and his/her colleagues examined bacterial adhesion process on polypropylene (PL) and expanded polytetrafluoroethylene (ePTFE) biomaterial surfaces. These two biomaterials are the widely used biomaterials in clinical practice. PL is macroporous material (1-2 mm), this biomaterial permits the passage of host defense cells such as polymorphonuclear cells or macrophages. Further, Law et al. (1994) demonstrated its tissue integration in the presence of bacterial contamination without alteration to the biomechanical resistance of the implant. When microorganisms settle in the internodal areas of the ePTFE implant, this infection may be extremely resistant to treatment since it is difficult for defense agents to reach these sites. In conclusion, the present findings indicate that Staphylococcus aureus and Staphylococcus epidermidis colonize the cross-over regions of the PL filaments, whereas in ePTFE prostheses it is the internodal areas which are mostly affected. The latter areas are of difficult access to defense agents.

Figure: Formation of a typical biofilm (B) on a polypropylene filament.

What is the source of the infecting agents? ^[8]

Bacteria happen to locate near implanted device either by contamination during implantation or by hematogenous seeding from a distant site. Airborne microorganisms, which can be present in the operating theater, can reach the surface as early as before implantation. Thus bacteria have chance to interact with bare substratum and if prevailed by attractive forces adhere to the surface tightly. Also during insertion of the biomaterial, microorganisms from the skin can adhere to it.

There is another pathway which is rather interesting. It has been proposed that macrophages play a role in transporting microorganisms to the biomaterials surface as some strains are capable to survive within macrophages. As the biomaterials surface elicits a foreign body reaction in the first weeks after implantation, and in the case of chronic inflammation also hereafter, macrophages are specifically attracted to the biomaterials surface thus potentially transporting microorganism to the biomaterials implant. As hematogenous infection can happen anytime, biomaterial implants are sometimes called “microbial time bombs”.

About half of all biomaterial-centered infections occur months to even years after deep tissue implantation which is termed as late BCI. There exists controversy concerning the origin of the infecting microorganisms in these late infections. There is no sufficient evidence for whether implants actually become infected through hematogenous path or whether late infections are caused by delayed appearance of perioperatively introduced bacteria.

Gottenbos et al. concluded in their study of late hematogenous infections of biomaterial in rats that late BCI of subcutaneous biomaterial does no occur in these animals. They have determined the susceptibility of subcutaneously implanted biomaterials to late hematogenous infection 4 weeks after biomaterial implantation. Two routes of hematogenous spreading of bacteria were used: either single intravenous injection of bacteria as a model for transient bacteremia, or by promoted bacterial translocation from the digestive tract. Bacterial translocation (BT) is the escape of mainly Gram-negative rods through the intestinal wall. BT can be promoted by nutritional factors, like total parenteral nutrition, fluid elemental nutrition, protein malnutrition, vitamin A deficiency and et c. Interestingly, also intraperitoneal implants promote BT.

It was found that none of the respective biomaterial discs in the non-septic rats became infected by intravenously injected bacterial strains. Five biomaterial discs revealed bacteria that were probably originating from perioperative contamination. The 4 infected biomaterial discs in the increased BT group showed exclusively staphylococcal strains, suggesting that translocation from the intestinal tract was not the source of infection, since staphylococci are numerous on the skin, but scarcely found in the gut flora. 

Clinical reports, on the other hand, show that in most hematogenous infections that are about 56 %, staphylococci are involved. They are the normal skin flora member of the man. The route here for bacteria might be the infected skin lesions producing relapsing bacteremia. Also dental or other surgical interventions, bacteriuria, intestinal surgery or pneumonia have been proposed as possible causes of hematogenous spreading of bacteria.

Hematogenous BCI can be induced directly after implantation, but are much more difficult to achieve after prolonged period of time. Essentially, the occurrence of BCI is a race for the surface. When infecting organisms arrive long-time after implantation on a biomaterial surface, the race is won in most cases by host cells and the biomaterial surface is out of reach for adhering organisms. Yet, many late biomaterial centered infections in man, most notably those associated with orthopedic implants are said to be hematogenous in origin, although it is also suggested that the hematogenous route of infection will only occur in immuno-compromised patients.

It seems that the evident difference in late infections in man and rats is caused by differences in the immune system of these species. Nonetheless, the case is difficult to resolve. The rationale behind the development of antimicrobial biomaterial surfaces considering late hematogenous BCI, I think should be the following: the surface should have such properties that would both form conditioning film and bind to bacteria readily.

Conclusion

            Biomaterial centered infection is one of the major problems in biomaterials science. In order to cope with the problem there used a series of different approaches. All of the approaches mainly depend on coatings that stay there for long time and leachable materials that is flow out of the biomaterial continuously which is eventually stops. Here we did not consider the sterilization procedures. It is out of scope of this paper.

            Although leachable materials such as antibiotics are sufficiently effective means of prevention BCI, they have the disadvantage of being transient. And therefore after some time, the implant should be removed and impregnated with the same chemical again. However, it is not practical and if not impossible at all. Moreover, every surgery or involvement means pain to the patient and potential contamination. Thus the permanent solution of coating seems to me as more practical solution. The approach of ‘biomimetics’ seemed to me very efficient and ideal one, though I cannot find any scientific papers concerning this method. Particle bombardment is promising method I think. Coating of polymers by classical polymerization procedures is well studied and used bur has the disadvantage of decreasing biomaterial integrity.

            The rationale behind designing biomaterial surface concerning its charge should be as following: bacteria should be able to bind to the surface but not proliferate. That is slightly positively charged surfaces seems to suit well the purpose. The main purpose in designing the surface of the material property should be the prevention of biofilm formation especially for completely buried implants.

References

[1]      Jucker BA, Harms H, Zehnder AJB. (1996). Adhesion of the Positively Charged Bacterium Stenotrophomonas (Xanthamonas) maltophilia 70401 to Glass Teflon. Journal of Bacteriology 178: 5472-5479.

[2]      Gottenbos B, Grijmpa DW, Van der Mei HC, Feijen J, Busscher HJ. (2001). Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria. Journal of Antimicrobial Chemotherapy 48: 7-13.

[3]      Multanen M, Talja M, Hallanvuo S, Siitonen A et al. (2000). Bacterial adherence to silver nitrate coated poly-L-lactic acid urological stents in vitro. Urological Research. 28: 327-331.

[4]      Multanen M, Talja M, Tammela TLJ, Seppala J et al. (2001). Biocompatibility of silver nitrate and ofloxacine coated bioadsorbable SR-PLLA rods. Urological Research. 29: 113-117.

[5]      Bos R, van der Mei HC, Busscher JH et al. (1999). Physico-chemistry of initial microbial adhesive interactions – its mechanisms and method for study. FEMS Microbiology Reviews. 23: 179-230.

[6]     Bellon JM, Natalio G-Honduvilla, Francisca Jurado, Alberto G-Carranza, Julia Bujan. (2001) In vitro interaction of bacteria with polypropylene/ePTFE prostheses. Biomaterials. 22: 2021-2024.

[7]      Gottenbos B, van der Mei HC, Klatter F, Nieuwenhuis P, Busscher HJ. (2002) In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber. Biomaterials. 23: 1417-1423.

[8]      Gottenbos B, Klatter F, van der Mei HC, Busscher HJ, Nieuwenhuis P. (2001) Late Hematogenous Infection of Subcutaneous Implants in Rats. Clinical and Diagnostic Laboratory Immunology. 8: 980-983.

[9]      Gorman SP, Jones DS. (2002) Antimicrobial Biomaterials for Medical Devices. A report. Medical Devices Group, School of Pharmacy, Queen’s University Belfast.

[10]   Flemming RG, Capelli CC, Cooper SL, Proctor RA. (2000) Bacterial colonization of functionalized polyurethanes. Biomaterials. 21: 273-281

[11]   Davenas J, Thevenard P, Philippe F, Arnaud MN. (2002). Surface implantation treatments to prevent infection complications in short term devices. Biomolecular Engineering. 19: 263-268