Disinfection of contaminated metal implants with an Er:YAG laser

Abstract Infections related to orthopedic procedures are considered particularly severe when implantation materials are used, because effective treatments for biofilm removal are lacking. In this study, the relatively new approach for infection control by using an erbium:yttrium‐aluminum‐garnet (Er:YAG) laser was tested. This laser vaporizes all water containing cells in a very effective, precise, and predictable manner and results in only minimal thermal damage. For preliminary testing, 42 steel plates and 42 pins were seeded with mixed cultures. First, the minimally necessary laser energy for biofilm removal was determined. Subsequently, the effectiveness of biofilm removal with the Er:YAG laser and the cleansing of the metal implants with octenidine‐soaked gauze was compared. Then, we compared the effectiveness of biofilm removal on 207 steel pins from 41 patients directly after explantation. Sonication and scanning electron microscopy were used for analysis. Laser fluences exceeding 2.8  J/cm2 caused a complete extinction of all living cells by a single‐laser impulse. Cleansing with octenidine‐soaked gauze and irradiation with the Er:YAG laser are both thoroughly effective when applied to seeded pins. In contrast, when explanted pins with fully developed biofilms were analyzed, we found a significant advantage of the laser procedure. The Er:YAG laser offers a secure, complete, and nontoxic eradication of all kinds of pathogens from metal implants without damaging the implant and without the possible development of resistance. The precise noncontact removal of adjacent tissue is a decisive advantage over conventional disinfectants. Therefore, laser irradiation could become a valuable method in every debridement, antibiotics, and implant retention procedure.


| INTRODUCTION
Infections represent the most common complication after surgical procedures and are of special concern when alloplastic implants are used. To date, the removal of the infected implant is frequently the ultimate therapeutic option. However, this procedure is associated with additional operations and commonly an inferior outcome for the affected patient.
Implant-related infections are usually caused by microorganisms that form biofilms. 1,2 Within biofilms, microorganisms are enclosed in a polymeric matrix and develop into complex communities, resembling multicellular organisms. 3 The biofilm shields the bacteria from host immune responses and from antimicrobial agents or antibiotics.
Reports in the literature indicate that 500 to 5000 times higher levels of antibiotics are needed to achieve the same antimicrobial effects on biofilm bacteria than are needed for planktonic bacteria. [4][5][6][7] To prevent implant-related infections, several alternative strategies have been tested in the last decade. These studies have investigated the bactericidal effects of silver and antibiotics used as coatings for metal implants [8][9][10][11][12][13] but showed elevated blood levels of silver ions, 8 clinical failure, 10 and even the development of bacterial resistance. [11][12][13][14] A relatively new approach to the treatment of microbial infections has been developed by dental surgeons, who use laser irradiation

| METHODS
Level of Evidence: II-a.
We used an in vitro approach with seeded metal implants as well as an ex vivo approach with contaminated half pins from extracted external fixators. External fixators gained wide acceptance in the treatment of open and juvenile fractures, in polytraumatised patients or deformity correction. 15 They were used for investigation because its pin sites are especially prone to infection, since the permanent skin wound facilitates the biofilm formation around the metal surface. Reported rates of pin-site infection vary widely in the literature, ranging from virtually zero to considerably over 50%, and may cause local infections and pin loosening but also osteomyelitis or sepsis. [16][17][18][19][20][21] For laser irradiation, we used a Burane Er:YAG laser (Wave Light, Germany) with a maximum energy of 2000 mJ and a maximum power of 20 W. The laser beam was used in the slightly defocused mode (spot size 3 mm diameter), applying 1600 mJ at a frequency of 8 Hz.
The fluence of the ablative pulses was 22.8 J/cm 2 . These parameters are the result of our preliminary findings, the rise in temperature within the pins, and the well-known predictable ability of tissue removal of about 2.5 μm/pulse/J/cm 2 and the collateral thermal tissue damage of about 20 µm. 22 Then, they were covered with tryptic soy broth (TSB) medium and cultivated for 2 weeks at 100 rpm in well titer plates with daily replenishment of the growth medium. For scanning electron microscopy (SEM) examination, the centers of another 14 seeded plates were irradiated with a single-laser impulse also using increasing pulse energies (0.2-2 J). These specimens were fixed in formaldehyde 7.5% and prepared for SEM according to a standard procedure. 24 KRIECHBAUMER ET AL.

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Fourty-two in vitro-seeded steel pins were divided into four groups and treated with the Er:YAG laser (1.6 J, spot size of 3 mm) either vertically (90°) or inclined (45°), cleaned mechanically with gauze soaked in octenidine (Octenisept, 0.1 g octenidine dihydrochloride and 2 g 2-phenoxyethanol, Schülke&Mayr Ges. m. b. H., Vienna, Austria) or kept untreated as control. Octenidine was used because of its broad spectrum antimicrobial effects against both Gram-positive and Gram-negative bacteria and fungi, 25,26 the resistance to blood and albumin, 27 its constant efficiency in the presence of organic matter, 28 and the low allergic potential. 26 For microbiological assessment, the laser was applied all over, whereas for SEM investigation, it was applied just in a single line along the pin to see the difference in the original and the lasered biofilm area.
Because potential collateral thermal damage is of concern in a surgical setting, additional sterile steel pins (diameters 3, 4, and 5 mm) were used to measure the rise of temperature during the laser irradiation process at different laser energies with an infrared thermometer (Exergen Temporal Scanner Infrared-TAT 5000; Exergen, Watertown, MA). In all laser experiments, no cooling devices (air, air-water spray, or flushing) were used.
We also tested the effect of the Er:YAG laser on 14 titanium plates (sectioned from PHILOS plates; DePuy Synthes) and observed that in contrast to steel implants, a macroscopic change of color occurred when very high-power settings (203 J/cm 2 ) were used.
These high-power settings were achieved by decreasing the spot diameter to 1 mm. This change of color is a well-known process for titanium and titanium alloys and is called an annealing process. This process results from the application of laser light to the surface, which causes local heating and oxidation of the metal, whereby oxygen is absorbed from the air. 29,30 We investigated if this thin oxide layer modifies the surface topography, the adhesion ability, or growth kinetics of microorganisms. Therefore, the titanium plates were irradiated with the Er:YAG laser at intermediate (22.8 J/cm 2 ) or very high (203 J/cm 2 ) power settings or kept untreated as control, and, subsequently, seeded with two different biofilm building bacteria (S. aureus and S. epidermidis) for 3 weeks. We evaluated the titanium surface morphology after annealing and surveyed the bacterial attachment and proliferation to this surface by SEM and sonication. In addition, we used SEM examination to visualize the induced biofilm removal. Therefore, the laser was applied just in a single line along the pin.

| Ex vivo experiments using Er:YAG laser disinfection
On the day when the external fixator was removed, the pin sites were clinically evaluated according to the pin grading system proposed by Clint et al, 32 which is based on three variables (erythema, discharge, and pain) and comprises three grades, named "good," "bad," and "ugly." Independent and experienced surgeons performed this evaluation and they divided the extracted half pins into three groups with the only constraints being that the overall amount of metal and pins proximally and distally to the fracture had to be evenly distributed. In addition, they took swab cultures from the pin sites of the control group.  On the in vitro-seeded pins, the biofilms in the control group contained an average of 1.96 × 10 6 CFU of bacteria and 3 × 10 3 CFU of yeast. Swab cleaning with octenidine achieved a complete eradication of bacterial and yeast colonization. Similarly, we did not see any remaining CFU after laser irradiation, when the pins were exposed to the laser beam (1.6 J) in a vertical manner. However, we found viable bacteria in two-thirds of the pins when the laser beam struck the metal at a 45°angle ( Figure 4). Because laser irradiation induces a temperature increase, pins of different diameters were measured with an infrared thermometer after single circumferential irradiation with different pulse energies and pulse repetition frequencies. As expected, higher repetition frequencies and pulse energies combined with small pin diameters lead to stronger heating. The highest temperature increase measured was +14.5°C in a 3 mm pin diameter at 2 J and 12 Hz ( Figure 5).
During the examination of the titanium plates, annealing was only observed at very high-power settings (>200 J/cm 2 ), and we typically noted a change of the original blue color to brown, which is the first annealing color in order from lowest temperature to highest.
However, the application of the Er:YAG laser with the previously described properties did not appear to have an effect on the microstructure or biocompatibility of the titanium surfaces ( Figure 6).
Particularly, we did not observe an increased biofim formation after annealing, since all plates were evenly seeded with microbes (S. aureus 5 × 10 3 CFU and S. epidermidis 3 × 10 6 CFU)

| Ex vivo experiments using Er:YAG laser disinfection
We analyzed a total of 207 pins after their extraction from 43 external fixators from 41 patients. The external fixators were removed after a median time period of 7.3 weeks (IQR 3-11). Seventy percent of pin sites were graded as "good," 23% as "bad," and 7% as "ugly" (Table 1).
Within the 20 different microbes identified in the biofilms from explanted pins, S. epidermidis was the most frequent bacteria associated with half pins (48.8%) and was detected in 41.6% of all patients showing signs of infection (pins graded "bad" or "ugly").
Staphylococcus haemolyticus and S. aureus were detected in 33.3% and 25% of all clinically infected patients, respectively.
The individual clinical assessment of the pins showed a significant correlation with the detected bacterial load identified (r = .398; P = .012). Pins rated as "good" showed a mean bacterial load of 6 CFUs (IQR 0-77), those rated as "bad" 75 CFUs (IQR 11-100), and those rated as "ugly" 181 CFUs .   The destructive effect of the Er:YAG laser derives from the spontaneous heating and evaporation of water that is part of every organism. However, heat is a double-edged sword that has a distinct bactericidal or bacteriostatic effect but may also harm the surrounding tissue. If used in a clinical setting, some thermal damage to the surrounding tissue cannot be avoided (direct irradiation, reflection, and heating of the implant). It is difficult to say how much collateral thermal damage can be tolerated by the surrounding tissue without scarring, but according to Ross et al, 36 it is not more than 300 µm in the perioral skin area. Even if skin is completely dissected with the Er:YAG laser, the thermal damage is restricted to a maximum of 210 µm, which makes scarring very unlikely. 37 In this study, no pin was heated to temperatures critical for bone necrosis (50°C for 1 minute). 38 Therefore, adverse effects on bone metabolism due to Er:YAG laser treatment are unlikely. In our setting, the temperature upshift of the implant was also not high (48°C) or long (10 min) enough to affect growth kinetics of the bacterial strains negatively. 39

| CONCLUSION
This study showed that the Er:YAG laser allows simple and complete biofilm removal from metal implants without the risk of harming patient or implant. The method works regardless of which pathogens are involved. This is of particular importance because we are increasingly confronted with resistance of microbes to pharmaceutical and chemical substances. We consider the Er:YAG laser a precise noncontact biofilm removal strategy producing an antiseptic wound surface without any further tissue damage and without the risk of resistance of microbes. According to these properties, laser irradiation could become a valuable method in every DAIR procedure and should therefore be further investigated.

ACKNOWLEDGMENTS
SEM has been performed at the University for Applied Science FH Technikum Wien and at CIUS Cell Imaging and Ultrastructural Research Unit, Vienna, Austria. This study is supported in part by the "Lorenz Böhler Fund" (Project Number 7/14).

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.