Introduction
Infection after intramedullary nailing of fractures is one of the most devastating situations in trau- ma surgery and can lead to septic non-union and chronic osteomyelitis [1, 2]. Infections can occur in closed fracture treatment [3] but open fractures in particular have a high risk of infection [4].
There has been a long controversial discussion whether open long bone fractures should be treated by a reamed or by an unreamed nail. The theoretical advantage of unreamed nails is the preservation of endosteal blood supply with a lower infection risk and better healing results [5].
However, reduction of infection rates by un- reamed nailing could never be proven and a re- cent systematic literature review showed that there were no differences in infection rates between un- reamed and reamed nailing [6]. In contrast to ini- tial hypotheses, several authors found that reamed nailing exhibited lower rates of non-unions, short- er fracture consolidation time and lower inci- dence of hardware failure [6±8] and advocated reamed nailing also for grade IIIB open fractures [9]. The use of implants for fracture treatment fa- vours infections compared to surgery where no foreign material is implanted into the body [10].
This is mainly related to the adhesion and prolif- eration of the bacteria on the implant surface [11], often with synthesis of an extracellular poly- saccharide matrix called biofilm (Fig. 7.3.1), which has been identified as a crucial step in the pathophysiology of infection and as an important factor of virulency of bacteria [10, 12]. This bio- film formation enables the bacteria to elude host defence and antibiotic treatment [13]. Staphylo- cocci are the most frequent infection-causing strains in bone and joint surgery [14]. Staph. epi- dermidis is found in up to 90% of all bone infec- tions with indwelling devices (osteosynthesis ma- terial, joint prostheses, etc.) [16], which is most likely related to the strong biofilm-building capac- ity of these bacteria [12]. Also certain strains of
Staph. aureus have been found to produce bio- films [16] and bone infections with Staph. aureus are in general more difficult to treat compared to infections with Staph. epidermidis [17]. Once bio- film formation is established, only implant re- moval promises eradication of the infection [18, 19]. Independently from the technique of intramed- ullary nailing, adequate prophylaxis is the most efficient way to prevent infection. Besides general pre-requirements such as aseptic operating room conditions, mainly soft-tissue management and the use of systemic antibiotics are crucial parame- ters to reduce infection rates [20]. A comprehen-
Plasma Polymer ± High-Porosity Silver Composite Coating for Infection Prophylaxis in Intramedullary Nailing
V. Alt, M. Wagener, D. Salz, T. Bechert, P. Steinrçcke, R. Schnettler
Fig. 7.3.1.
Scanning electron microscopy of biofilm-produc-
ing Staph. aureus.
aBiofilm formation of Staph. aureus on
the thread of a screw.
bHigher magnification reveals the
round staphylococci embedded in the biofilm. Magnifica-
tions:
a´83,
b´ 521. Source: Laboratory of Experimental
Trauma Surgery, Giessen, Germany
sive overview of intravenous antibiotic prophy- laxis for open fractures is given in Chap. 7.1.
Furthermore, coating of implants promises pre- vention of colonisation of the implant's surface with reduction of infection rates. Gentamicin is the most common antibiotic used for loading of bone cements in infection prophylaxis in total joint surgery in Europe [21] due to its good anti- bacterial activity, excellent biocompatibility, and favourable release kinetics [22]. Recently, gentami- cin was used in a poly(D,L-lactide) matrix for coating of osteosynthetic implants in an experi- mental study [23].
Also metallic silver in the form of high-porosi- ty silver particles was shown to exhibit high anti- microbial activity in bone cements including ac- tivity against multiresistant strains, e.g. methicil- lin-resistant Staph. aureus (MRSA) and methicil- lin-resistant Staph. epidermidis [24]. Metallic sil- ver can also be used in combination with a SiO
xplasma polymer for coating of osteosynthetic de- vices.
Plasma Polymerisation Coating Technique Plasma polymerisation is a versatile technique for the preparation of functional coatings on various materials and devices. Depending on the precur- sor material and the processing parameters, films with a thickness well below 100 nm can be ob- tained. In general these films are chemically inert, insoluble, mechanically and thermally stable and are used as membranes or protective coatings, for
example [25]. By using hexamethyldisiloxane (HMDSO) as a precursor material, it is possible to obtain highly biocompatible films due to the formation of a coating that basically consists of a SiO
xplasma polymer.
Plasma Polymers Containing Metal Particles Small metallic silver particles can be embedded into a SiO
xplasma polymer film in order to achieve an antimicrobial effect with such a coat- ing on orthopaedic implants. Plasma polymer ma- trices for the embedding of metal clusters were used for the first time by Perrin et al. [26]. The deposition technique developed by these authors is based on the simultaneous deposition of the metal by sputtering and plasma polymerisation.
An alternative method is the simultaneous deposi- tion of the metal by evaporation and plasma poly- merisation [27±31]. The coatings investigated in this work have been prepared by silver cluster de- position via inert gas evaporation and condensa- tion and subsequent plasma polymerisation. Two different types of coatings have been prepared:
for the coating named type A the silver clusters have been directly deposited on the substrate ma- terial and were embedded in a plasma polymer film, the coating named type B consists of silver clusters being embedded between two plasma polymer layers so that the silver is not in direct contact with the substrate material (Fig. 7.3.2).
Fig. 7.3.2.
Model of the two
different coatings prepared
in this work
Implants Coated with a
Plasma-Polymer-Containing Metallic Silver As test implant materials, stainless steel and tita- nium Kirschner wires with trocar points (diame- ter 2 mm, length 150 mm) were used. The metallic silver clusters were produced using an inert gas evaporation technique. Silver wire (Alfa Products, Karlsruhe, Germany; metal impurities less than 15 ppm) is evaporated from a resistance-heated tungsten crucible in an argon atmosphere at pres- sures between 5 and 10 mbar. The silver clusters are deposited on the substrate, which is rotated in the metal vapour to achieve a homogeneous silver cluster distribution on the substrate. The evapora- tion rate can be controlled by the current of the vapour source. The subsequent plasma polymer film was prepared in a microwave discharge from HMDSO monomers at 2500 Wand at a process gas flow between 20 and 400 sccm.
For the characterisation of the metal±plasma polymer composites, transmission electron micro- scopy (TEM) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS) were used. The TEM analysis of the composite coatings showed that the silver forms clusters with a size of ap- prox. 10 nm on the substrate materials (Fig.
7.3.3). The clusters form agglomerates, which are supposed to be porous instead of solid silver is- lands. Between the agglomerates of silver clusters open channels exist. Therefore, the plasma poly- mer film that has been deposited on top of the silver clusters has contact with the substrate mate- rial. That results in a good adhesion of the plas- ma polymer onto the substrate.
From the TOF-SIMS analysis (Fig. 7.3.4), it can be seen that the plasma polymer film is approxi-
mately 40 nm thick (5000 s of sputtering corre- sponds to 45 nm polymer film). Only Si and C can be seen during the first 5000 s of sputtering.
Below the polymer film, silver is found (see in- creasing intensity of Ag beyond 5000 s sputtering time). Not only can the principal structure of the film be confirmed by this method, but also the infiltration of the silver agglomerates by the plas- ma polymer film can be confirmed as the Si sig- nal does not change dramatically when the Ag signal increases. The structural analysis of the film by TEM and TOF-SIMS confirmed the mod- els shown in Fig. 7.3.2.
Antimicrobial Testing
For antimicrobial testing, an in vitro microplate proliferation test was used that has shown its ac- curacy for bone cement samples [32] and for bio- materials in general [33]. In brief, the samples are tested by three different subsequent incubation steps. During the first step, they are incubated in wells of a 96-well microplate with 10
6colony- forming units of bacteria per well in a 250-ll cell suspension for 1 h, allowing adhesion of the
Fig. 7.3.3.
Transmission electron microscopy of silver clus-
ters in a plasma polymer film
Fig. 7.3.4.TOF-SIMS analysis of silver clusters in a HMDSO
plasma polymer film (type A)
strain on the samples. After this first incubation, rinsing of the samples with PBS removes loosely attached cells from the sample surface. In a sec- ond step, the remaining adherent bacteria on the implant surface are incubated in minimal medium (PBS with 0.25% glucose, 0.2% (NH
4)
2SO
4, and 1% sterile trypticase soy broth) for 18 h in an- other 96-well microplate. If there is no antimicro- bial effect of the implant, attached cells on the surface of the sample start to multiply and to re- lease clonal counterparts into the well. If there is an antimicrobial effect of the implant, the cells are killed and are not able to seed out daughter cells. After removal of the samples the released bacteria were amplified in a final step by adding 50 ll of TSB medium to each well for 36 h, which can be analysed by a microplate reader (Versa- Max, Molecular Devices, Sunnyvale, California, USA) system providing time-proliferation curves for each tested sample. These curves allow differ- entiation between the absence of antimicrobial ac- tivity, intermediate antimicrobial, and bactericidal effects of the tested sample (Fig. 7.3.5).
The samples were tested against Staph. epider- midis, which is the most common strain in de- vice-associated infections [15].
Antimicrobial Activity of Silver-Coated Implants
Uncoated samples of both stainless steel and tita- nium wires showed uninhibited proliferation of the tested Staph. epidermidis indicating missing antimicrobial properties of the device (Fig. 7.3.6).
In contrary to this, coating of the material with high-porosity silver directly on the substrate (type A) showed no bacterial growth for stainless steel and titanium. This means that this coating type of osteosynthetic devices exhibits bactericidal ac- tivity both for stainless steel and titanium. If the metallic silver is embedded between two polymer layers (coating type B) on stainless steel there was a delayed onset of bacterial growth indicating in- termediate antimicrobial activity for this material- coating combination. However, coating type B ex- hibited also bactericidal effects on titanium im- plants.
In summary, coating with direct application of the metallic silver plasma polymer matrix onto the surface of implants showed very promising bactericidal activity on both stainless steel and ti- tanium.
Fig. 7.3.5.
Curve 1 uninhibited bacterial proliferation indi- cating missing antibacterial properties of the implant; curve 2 delayed onset of bacterial proliferation indicating slight
antibacterial effect of the implant; curve 3 inhibited bacteri-
al growth indicating bactericidal effect of the implant
Discussion and Conclusion
This investigation showed the high antibacterial activity of metallic silver in a SiO
xplasma poly- mer coating of osteosynthetic devices. In sum- mary, a suitable technique for metallic silver coat- ing of implants was found, which promises new prophylaxis options in fracture treatment includ- ing intramedullary nailing.
The antimicrobial activity of silver is based on different effects on the cells, e.g. binding to and subsequent inactivation of SH-groups of enzymes and other proteins [34, 35], interference with the energy production and energy conservation of the bacterial cell [36]. Silver does not prevent adhe- sion of the bacteria to the implant surface but kills adherent microorganisms and inhibits their proliferation [33]. This mechanism is the most likely reason for prevention of infection in silver- loaded devices, e.g. in catheters [37] or in bone cement [24]. If bacterial proliferation is inhibited by silver, the formation of biofilm will also be avoided.
Compared to gentamicin coating of implants, which has been used in another study with fa- vourable results against Staph. aureus, silver has
two distinct advantages. First, silver is effective against almost all strains including MRSA and MRSE [24] because silver resistance does not play an important role in the hospital microbial germ flora [38]. A possible explanation for this phe- nomenon is that silver acts on different metabolic levels in the cell and, therefore, resistance cannot be acquired by single-point mutation as for ami- noglycoside antibiotics like gentamicin. With in- creasing incidences of multiresistant strains in nosocomial infections, the broad antimicrobial ac- tivity of silver is a very favourable new option in infection prophylaxis. Second, even if infection occurs in silver-coated implants, there are still all options given for antibiotic treatment of the pa- tient. In cases in which gentamicin is used as in- fection prophylaxis, the infection-causing strains often acquire gentamicin resistance [39]. This gentamicin resistance is often combined with re- sistance against other aminoglycosides and ex- cludes the further use of these agents against the infection.
The metallic silver that was used for the coat- ing of the implants clearly differentiates from ªcommercial silver coatingsº. The silver is em- bedded in a plasma polymer film and forms a
Fig. 7.3.6.
