CN117320785A - Alternating magnetic field and antibiotic for eradicating biofilm on metals in a synergistic manner - Google Patents

Abstract

Metal-related infections such as artificial joint infections (PJI) lead to significant morbidity worldwide. Infected implants often require surgical removal and weeks of antibiotics. This is due in large part to the formation of biofilms. Embodiments described herein utilize Alternating Magnetic Fields (AMFs) as a non-invasive method for eradicating (i.e., significantly reducing) biofilms on metals. Embodiments employ short intermittent bursts of AMF administered in concert with traditional antibiotics to synergistically remove biofilm on metals. This effect is seen throughout the common PJI-associated pathogens and clinically used antibiotics. The use of AMF in an intermittent manner has important implications for providing a non-invasive treatment that is both safe and effective for the patient.

Description

Alternating magnetic field and antibiotic for eradicating biofilm on metals in a synergistic manner

The invention was completed with government support under AI155291 awarded by the national institutes of health (The National Institutes of Health) (NIH). The government has certain rights in the invention.

Cross-reference to related applications

The application claims priority from U.S. provisional patent application No.63/169,636, filed on 1/4/2021, and entitled "Alternating Magnetic Fields and Antibiotics to Eradicate Biofilm on Metal in a Synergistic Fashion," the contents of which are hereby incorporated by reference. The application also claims priority from U.S. provisional patent application No.63/325,298, filed 3/30/2022, entitled "Alternating Magnetic Fields and Antibiotics to Eradicate Biofilm on Metal in a Synergistic Fashion," the contents of which are hereby incorporated by reference.

Background

Alternating Magnetic Field (AMF) is a non-invasive method of treating implant-related infections.

Brief Description of Drawings

Features and advantages of embodiments of the present invention will become apparent from the appended claims, the following detailed description of one or more exemplary embodiments, and the corresponding figures. Where considered appropriate, reference numerals have been repeated among the figures to indicate corresponding or analogous elements.

Fig. 1A, 1B, and 1C discuss simulation and measurement of Intermittent Alternating Magnetic Field (iAMF) heating. The experimental set-up consisted of a stainless steel ring with biofilm in a medium in a 50-ml tube and held in place by a plastic scaffold (fig. 1A). The tube is placed in the solenoid coil (analog image in fig. 1A). The display of the iramf dosing regimen (fig. 1B), where the doses were separated by several hours (Δtdose, top panel). Each dose consists of a number of AMFs short-term exposures (Nexp) of AMFs delivered at intervals (Δtexp) for several seconds (texp, exposure time required to reach target temperature, tmax) followed by a temperature drop after AMF shutdown (fig. 1B, middle image). The temperature of the ring versus time when the AMF was exposed to Tmax at 50, 65 and 80 degrees celsius is shown (fig. 1C). Simulated AMF heating of metal rings for different exposure times depicts spatial temperature changes on the surface and minimal heating of surrounding medium. The mean and standard deviation of the temperatures are shown.

Figures 2A, 2B, 2C, 2D discuss how iAMF and ciprofloxacin synergistically reduce pseudomonas aeruginosa (p.aerosea) biofilm. (fig. 2A) general treatment regimen for combining iAMF and antibiotics. (FIGS. 2B, 2C, 2D) bacterial log reduction (log reduction) of PAO1 biofilm over a 24-hour period when treated with (B) 80 ℃, (C) 65 ℃ or (D) 50 ℃ different peak temperatures Tmax using iAMF heating alone (blue dotted line), 0.5mg/mL ciprofloxacin alone (black solid line) or iAMF+ciprofloxacin (blue solid line). As shown in the figure, the number of exposures is different for each case. Untreated controls (black dashed line) were not exposed to antibiotics or AMF. Colony Forming Units (CFU) were counted at 0, 12 hours (before and after AMF) and 24 hours after treatment. CFU detection Limit (LOD) =0.78 log (CFU/cm 2). Statistical significance: insignificant (ns) significance of p <0.0001 (/ x).

Figures 3A, 3B, 3C, 3D discuss how iAMF and ciprofloxacin cause changes in bacterial morphology. Laser scanning confocal microscopy of pseudomonas aeruginosa (PAO 1) biofilm-infected loops 12 hours after treatment began. Living bacteria within the biofilm express Green Fluorescent Protein (GFP), while EPS is stained with Concanavalin (A-Alexa Fluor 647 conjugate, thereby emitting red fluorescence. The rings (FIG. 3A) were treated with iAMF (Tmax=65℃) for 1 hour, then incubated in MHII medium for 12 hours, (FIG. 3B) in 0.5. Mu.g/mL ciprofloxacin or (FIG. 3C) with 1hiAMF during 12 hours of incubation with 0.5. Mu.g/mL ciprofloxacin. (FIG. 3D) untreated control. Scale bar: 100 μm.

FIG. 4 discusses how the combination of iAMF and ciprofloxacin shows a dose-dependent subtraction of P.aeruginosa biofilmFew. The iramf dose (tmax=65 ℃, Δtexp=5 minutes) was delivered at 0 hours and 12 hours, with 3, 6 or 12 exposures (simultaneous incubation with 0.5mg/mL ciprofloxacin) per dose. Colony Forming Units (CFU) were counted at time points of 0, 12 and 24 hours immediately after the first iAMF dose (left) and 24 hours after treatment (right). For treatment with ciprofloxacin alone (after the first dose without AMF), CFU were counted after 1 hour in ciprofloxacin. CFU at time 0 was 6.81log (CFU/cm ² ). CFU limit of detection (LOD) =0.78 log (CFU/cm) ² ). p=0.0318 and p<0.0001(****)。

Fig. 5A and 5B discuss how iAMF and antibiotics synergistically reduce staphylococcus aureus (s.aureus) biofilms. Staphylococcus aureus (UAMS 1) biofilms were treated with imaf doses (15 min/dose, tmax=65 ℃, Δtexp=5 min) and the indicated antibiotics for 0 and 12 hours. (FIG. 5A) logarithmic biofilm reduction (CFU) after 24 hours using iAMF and 2. Mu.g/mL ceftriaxone. CFU was calculated at time points 0, 12 and 24 hours. (FIG. 5B) CFU of Staphylococcus aureus biofilm 24 hours after treatment with iAMF and ceftriaxone (2. Mu.g/mL) or linezolid (2. Mu.g/mL). CFU detection Limit (LOD) =0.78 log (CFU/cm 2). Statistical significance: insignificant (ns) p=0.0004 (and) p <0.0001 (/ x >) significance.

Fig. 6A, 6B and 6C discuss how iAMF can reduce MDR pathogens depending on the resistance mechanism. MDR pseudomonas aeruginosa (MB 699) biofilms were treated with meropenem (MIC 64 μg/mL) or ciprofloxacin (MIC 64 μg/mL) with or without iAMF (dosing at 0 and 24 hours, nexp=12, tmax=65 ℃, dtexp=5 minutes) while incubated with antibiotics for 48 hours. Fig. 6A: a mechanism for sensitizing antibiotic resistant biofilms to meropenem by AMF. Fig. 6B: treatment time course with 64 μg/mL meropenem (left) or ciprofloxacin (right). Colony Forming Units (CFU) were counted at time points of 0, 24 and 48 hours. Fig. 6C: log reduction of antibiotic resistant biofilms at different concentrations of ciprofloxacin or meropenem 48 hours after treatment initiation. CFU detection Limit (LOD) =0.78 log (CFU/cm 2). Statistical significance: not significant (ns), p=0.0001 (x), and p <0.0001 (.

FIG. 7 discusses combined iAMF/antibiotic treatment of P.aeruginosa (PAO 1) biofilms grown on plastic and metal rings with 0.5 μg/mL ciprofloxacin. Biofilms were treated with iAMF dose (tmax=65 ℃, Δtexp=5 minutes for administration duration 1 hour) and 0.5 μg/mL ciprofloxacin at 0 hours. Colony Forming Units (CFU) were counted at 12 hours. CFU detection Limit (LOD) =0.78 log (CFU/cm 2). Statistical significance: insignificant (ns) and p <0.0001 (/ x).

Figure 8 discusses SEM images of antibiotic-resistant pseudomonas aeruginosa (MB 699) biofilms treated with imaf and antibiotics. With iAMF (N) _exp ＝12，T _max ＝65℃，Δt _exp =5 min) and incubated in 64 μg/mL meropenem or ciprofloxacin for 12 hours. Magnification factor: 35,000×. Scale bar = 300nm.

Fig. 9 includes physical properties of materials used for simulation.

Fig. 10 includes the iAMF parameters for different target temperatures (Tmax). * 80 degrees celsius is reached within 6 seconds after exposure and held near that temperature for an additional 6 seconds with Proportional Integral Derivative (PID) calibration before stopping the AMF.

FIG. 11 includes the minimum inhibitory concentration of antibiotics used to treat Pseudomonas aeruginosa strains.

Fig. 12 includes a protocol or method in an embodiment.

Fig. 13 includes a method in an embodiment.

Figures 14, 15 and 16 include systems for implementing embodiments.

Fig. 17 includes signal characteristics of burst exposure and signal characteristics of thermal exposure in an embodiment.

Fig. 18 includes CEM43 measurements around the ring during the iAMF. Assuming the ring is surrounded by muscle tissue, simulations were performed to calculate CEM43 at different distances from the ring using the iAMF. Three iAMF treatment conditions were used: nexp=1, tmax=80 ℃; nexp=1, tmax=65 ℃; nexp=12, tmax=65 ℃, dtexp=5 minutes. 240 minutes represents the threshold for irreversible cell damage.

Fig. 19 includes the heat treatment time of the biofilm and FIC index of ciprofloxacin concentration. PAO1 biofilms were treated at time 0 at 65℃for a period of time and incubated with ciprofloxacin at various concentrations for 12 hours or 24 hours at 37 ℃. The numbers in the heat map (heat map) show the FIC index values of the treatment combinations. FIC values less than or equal to 0.5 are considered synergistic, values >0.5 and <4 indicate no interaction or additivity, and values greater than or equal to 4 indicate antagonism. n=3.

FIGS. 20A, 20B, 20C, 20D show that iAMF and antibiotics can act on biofilms of various lifetimes. Pseudomonas aeruginosa (PAO 1) and Staphylococcus aureus (UAMS 1) biofilms were cultured according to the same protocol with medium supplementation every 24 hours until day 7. The biofilm was then treated with imaf doses (tmax=65 ℃, Δtexp=5 minutes per dose, 15 minutes) and the indicated antibiotics at 0 and 12 hours. (FIG. 20A) 7-day P.aeruginosa (PAO 1) biofilm log reduction (CFU) after 24 hours using iAMF and 0.5 μg mL-1 ciprofloxacin. CFUs were counted at time points 0, 12 (before and after AMF) and 24 hours. (FIG. 20B) 7-day Staphylococcus aureus (UAMS 1) biofilm log reduction (CFU) after 24 hours using iAMF and 2. Mu.g mL-1 linezolid. CFU was calculated at time points 0, 12 and 24 hours. (fig. 20C) comparison of previous 2-day (48 hours) and 7-day pseudomonas aeruginosa (PAO 1) biofilms at times 0 and 24 hours with the same iAMF (tmax=65 ℃, Δtexp=5 minutes, 15 minutes per dose) treatment and 0.5 μg ml-1 ciprofloxacin. (fig. 20D) comparison of 2-day (48 hours) and 7-day staphylococcus aureus (UAMS 1) biofilms at times 0 and 24 hours with the same iAMF (tmax=65 ℃, Δtexp=5 minutes per dose, 15 minutes) treatment and 2 μg mL-1 linezolid. n=3. Error bars (error bars) represent SD. CFU limit of detection (LOD) =0.78 log (cfum cm-2). Two-factor analysis of variance. Statistical significance: not significant (ns).

Detailed description of the preferred embodiments

Reference will now be made to the drawings wherein like structures may be provided with like suffix reference designations. In order to more clearly illustrate the structure of the various embodiments, the drawings included herein are graphical representations of the structure. Thus, the actual appearance of the manufactured structure (e.g., in a photograph) may appear to be different, but still incorporate the claimed structure of the illustrated embodiment (e.g., in an actual manufactured device, the walls may not be entirely perpendicular to each other). Furthermore, the drawings may show only the structures for understanding the illustrated embodiments. Additional structures known in the art may not have been included to maintain the clarity of the drawing. For example, not every layer of the device is necessarily displayed. "one embodiment," "various embodiments," etc., indicate that one or more embodiments so described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Some embodiments may have some, all, or none of the features described for other embodiments. "first," "second," "third," etc. describe a common object and indicate different instances of like objects mentioned. Such adjectives do not imply that the objects so described must be in a given sequence, whether temporally, spatially, in ranking, or in any other manner. "connected" may indicate that elements are in direct physical or electrical contact with each other, and "coupled" may indicate that elements co-operate or interact with each other, but they may or may not be in direct physical or electrical contact. Phrases such as "including at least one of a or B" include cases with A, B or a and B.

Evidence a is referenced in the previously filed provisional patent application. The subject matter from evidence a is now directly included in the present specification. The topic from evidence a is followed by a discussion entitled "further discussion of embodiments". The references discussed in the discussion of evidence a below are at the end of the written description of the specification.

Evidence A

Discussion of embodiments a non-invasive intermittent alternating magnetic field in combination with an antibiotic that reduces metal-related biofilms in a synergistic manner.

Several hundred thousand human implants each year require surgical repair due to infection. It is difficult to treat infections with conventional antibiotics due to the formation of biofilms on the implant surface. Embodiments discussed herein include a non-invasive method of using an Intermittent Alternating Magnetic Field (iAMF) to eliminate biofilm on a metal implant. As used herein, "eliminate" does not necessarily mean, for example, a complete 100% removal or rupture of the biofilm, but may mean, for example, a significant reduction of the biofilm. Embodiments demonstrate that iAMF and an antibiotic have a synergistic effect in their biofilm reducing ability. For pseudomonas aeruginosa biofilms, bacterial load was reduced by >3 log (p < 0.0001) using iAMF and ciprofloxacin after 24 hours compared to either treatment alone. The effect is not limited by pathogens or antibiotics, as similar biofilm reduction is observed with imaf and linezolid or ceftriaxone in staphylococcus aureus. The imaf and antibiotic efficacy are visible throughout various imaf settings, including different imaf target temperatures, dose durations, and dosing intervals. Preliminary mechanism studies revealed that membrane rupture is a factor important for the enhanced antibacterial activity of AMF in the biofilm context. Embodiments demonstrate the efficacy of using a non-invasive method to reduce biofilm on metal implants.

Introduction to evidence A

Metallic implants such as artificial joints, bone fixation hardware, and dental implants are widely used in medicine to replace damaged or diseased tissue (reference 1). Millions of metal devices are implanted into people every year in total worldwide (reference 2). In the case of Total Knee Arthroplasty (TKA), more than 100 tens of thousands of procedures are performed annually in the united states, and the number is expected to reach-350 tens of thousands by 2030 due to population and health trends (reference 3). About 1-2% of these implants are infected. The serious complications are challenging to treat (reference 3). Currently, the treatment of Prosthetic Joint Infections (PJI) relies primarily on multiple revision surgeries. An initial surgical procedure was performed to remove the infected implant and to place a temporary spacer (reference 4). Antibiotics are administered for several weeks to clear residual infection. Once it is confirmed that the patient is not infected, a final surgical operation is performed to implant a new prosthesis (reference 5). Treatment of PJI is highly invasive, with significant negative impact on the quality of life of the patient. Furthermore, the failure rate of these multi-stage surgical procedures is currently more than 10% (references 6, 7). Furthermore, the cost of treating PJI, which was expected in the united states alone in 2020, was $ 16 billion, creating a significant economic burden on the health care system (reference 8).

The main reason for the inefficiency of antibiotic treatment of metallic implant infections such as PJI is due to the formation of biofilm on the implant surface (reference 9). Biofilms are thin (tens to hundreds of microns) aggregates of bacteria and Extracellular Polymeric Substances (EPS) (reference 10). EPS is produced by bacteria and forms a barrier to the surrounding environment, thereby making these organisms up to thousand times more resistant to antibiotics and providing protection from the immune system (reference 11). Importantly, the increasing antibiotic resistance only further complicates the problem. In addition to PJI, biofilms play an important role in infections of other widely used medical implants, including catheters, mechanical heart valves, and bone fixation hardware (references 1, 12, 13).

A non-surgical means of eradicating (i.e., significantly reducing) the biofilm would be a significant advancement in the treatment of Metal Implant Infections (MII). Several physical methods for eliminating (i.e., significantly reducing) biofilms have been proposed, including electrical current (references 14-16), ultrasound (reference 17), heat (references 18-20), and shock waves (reference 21). However, these methods are either difficult to apply in vivo or have limitations for use on metal implants. One potentially safer and more effective method of metal implant biofilm removal is through the use of AMF. AMFs can be delivered from outside the body and are not damaged by penetration depth limitations or complex wave distortion across tissue boundaries. When a metal implant is exposed to AMF, a current is induced on the surface, resulting in the generation of heat. Previous studies have shown the feasibility and effectiveness of biofilm elimination (e.g., significant reduction) by AMF (references 19, 22). After only a few minutes of AMF treatment, the biofilm on the stainless steel gasket (washer) was significantly reduced (reference 22).

However, applicants have determined that the necessity of maintaining temperatures in the range of 50-80 degrees celsius for several minutes to achieve biofilm reduction presents challenges for AMFs to be clinically used. Furthermore, incomplete eradication of bacteria via AMF resulted in regrowth within a short period of time (reference 22). Embodiments include a method of overcoming the disorder, i.e., combination therapy with an antibiotic. In vitro studies have shown a greater and sustained reduction in bacterial load. Thus, applicants determined that combined AMF and ciprofloxacin were observed to reduce biofilm more effectively than AMF or ciprofloxacin alone and prevented recurrence up to 24 hours post-treatment (references 20, 23, 24). Furthermore, applicants have noted that the use of a brief, intermittent AMF exposure can address the problems of elevated implant temperature and safety. As previously shown in the murine model, applicants noted that rapidly raising the metal implant to the target temperature and for a short period of time resulted in much less tissue damage than longer duration exposure (reference 25). Further, applicants note that these short duration exposures can be repeatedly delivered with sufficient cooling time between exposures to allow for therapeutic thermal dosing on the implant surface without concomitant increases in tissue thermal dose. The process is called batch AMF, or iAMF.

Embodiments include the efficacy of the iAMF exposure in combination with an antibiotic to eliminate (i.e., significantly reduce) biofilm on metal surfaces in vitro. Applicants determined the relationship between AMF parameters (temperature, duration, number of exposures) and antibiotics (drug, concentration, administration). Applicants explored the method in both prototype gram-positive and gram-negative pathogens and explored the mechanisms underlying the mechanistic relationships by attempting to reduce multi-drug resistant pathogens with iAMF.

Evidence A results

The iAMF exposure is generated using an in vitro system designed to heat the metal ring for a precisely controlled exposure duration and specified exposure and dosing intervals. The system consists of 32 identical solenoid coils capable of producing a uniform AMF (10.2±0.3 mT) in the center of each coil. Furthermore, the measured magnetic field is very consistent with the predictions from the simulation (11.2±0.4 mT). The metal rings are chosen because they are expected to heat uniformly in the magnetic field of the solenoid when oriented along the coil axis, as shown in fig. 1A. The finite element simulation results in fig. 1C confirm the uniform heating achieved. The surface temperature distribution on the ring after 1.2, 3 and 6 seconds of heating is shown with a uniform temperature around the circumference of the ring and a standard deviation between the top and middle of no more than 2 degrees celsius. Further, the simulation underscores that for these short durations of heating, the medium surrounding the ring is not significantly heated, which is also observed by practical measurements. Cumulative equivalent minutes at 43 ℃ (Cumulative equivalent minutes) (CEM 43) was used to evaluate mammalian cell thermal injury (reference 26). Typically 240 minutes is considered to be the threshold for permanent damage to muscle tissue (references 27, 28). Because heat transfer from the ring to the adjacent medium is controlled by thermal conduction and convection, applicants calculated CEM43 around the ring assuming that the ring is surrounded by muscle tissue (i.e., only thermally conductive). CEM43 was no more than 240 minutes at 2mm from the ring in the case of imaf at tmax=80 ℃ and at 1mm in the case of 12 imaf exposures at tmax=65 ℃, suggesting no permanent tissue damage at that distance (fig. 18).

After characterizing the kinetics of ring heating using the iAMF system, applicants studied their ability to eradicate biofilm from the ring surface (fig. 2A-2D). As used herein, "eradicating" does not necessarily mean, for example, complete 100% removal or rupture of the biofilm, but may mean, for example, a significant reduction of the biofilm. Each of the three iAMF treatments studied (blue dashed line) was able to reduce the pseudomonas aeruginosa PAO1 biofilm by about 1-2 log after each dose. However, between doses, CFU levels returned to baseline. The ring exposed to 0.5ug/ml ciprofloxacin alone (solid black line) showed a stable CFU reduction of almost 3 log over the first 12 hours, followed by plateau. Remarkably, the iAMF exposure in combination with ciprofloxacin (blue solid line) showed unexpected results, namely a consistent reduction of the biofilm to the detection limit. The decrease in CFU immediately after each dose was equal or greater for the combination treatment compared to the iAMF alone. Between the AMF doses at time 0 and 12 hours, there was a further decrease in CFU, probably because ciprofloxacin showed enhanced activity in the biofilm. Notably, the magnitude of CFU reductions at 0 and 12 hours were similar, suggesting a consistent AMF treatment effect after each dose. The trend was observed for three different treatment strategies that varied target temperature (Tmax) and number of exposures (Nexp). In addition, more exposure was required at lower temperatures to observe equivalent reductions in biofilm after 2 doses (fig. 2B, 2C, 2D). At 24 hours, the differences in CFU between the combined treatment group and all other groups were very significant (p < 0.0001). The same treatment strategy with the combination of imaf and ciprofloxacin at tmax=65 degrees celsius was performed on the same size plastic ring or grade 5 titanium ring with pseudomonas aeruginosa biofilm. On plastic rings, biofilm CFU showed no significant differences when treated with iAMF and ciprofloxacin compared to ciprofloxacin alone incubation (fig. 7). For biofilms on titanium rings (a material widely used for medical implants), the decrease in the biofilm for the iAMF and ciprofloxacin treatments was similar to that seen on stainless steel rings.

To evaluate whether there is a synergistic relationship between heat and antibiotics on the biofilm, experiments were performed using a temperature controlled water bath. The biofilm was exposed to different durations of heating at the indicated temperatures, and then CFU reduction in bacteria with and without various antibiotic concentrations was quantified (see supplementary material). MBEC (minimum biofilm eradication concentration) was used to quantitatively study the synergistic effect of heat and ciprofloxacin, as previously described (reference 29). The results show synergy (references 30, 31) with partial inhibitory concentration (FIC) index values (definition of synergy) below 0.5 for various combinations of heat treatment time and ciprofloxacin concentration at both 12 hours and 24 hours after a single heat treatment. This suggests that heat and ciprofloxacin show synergistic activity in the biofilm background (fig. 19) (reference 29).

Enhanced reduction in biofilm for the combined imaf and antibiotic was also visually observed using laser scanning confocal microscopy (fig. 3A-3D). GFP-PAO1 biofilms were treated with imaf (tmax=65 degrees celsius, Δtexp=5 minutes, nexp=12) and 0.5 μg/mL ciprofloxacin. GFP-PAO1 cells were shown green and concanavalin A-AlexaFluor647 stained EPS was shown red. This allows the morphology of the bacterial cells to be observed under different processing conditions. In the case of ciprofloxacin only (fig. 3B), the bacteria showed a slight elongation at 12 hours after treatment compared to the imaf only (fig. 3B) and the control (fig. 3D). Although only the group of iAMF showed diffuse concanavalin A-Alexa Fluor647 stained EPS, the combined treatment of iAMF and ciprofloxacin (fig. 3C) had less concentrated EPS staining. Furthermore, there was an increased number of elongated GFP-expressing cells, a visual indication of pseudomonas during quinolone treatment (references 32, 33).

The effect of the iAMF dose duration was studied in more detail. The pseudomonas aeruginosa biofilm was treated with iAMF (tmax=65℃) for a dosing duration ranging from 15 minutes to 1 hour in combination with 0.5 μg/mL ciprofloxacin according to the same treatment protocol as in fig. 2A. The exposure interval was 5 minutes in each treatment. Immediately after combined iAMF and antibiotic treatment, the decrease in CFU showed a dose dependent response, with longer iAMF duration resulting in greater decrease (fig. 4, p=0.0318 for 15 min iAMF, and p <0.0001 for 30 and 60 min iAMF). After 15 minutes of iAMF, there was a 1.41 log reduction, which increased to a 2.68 log reduction after 1 hour of dosing. After 24 hours, there was a 2.7 log reduction in biofilm treated with ciprofloxacin alone, while combination treatment achieved a greater than 5 log reduction, approaching the limit of detection for all iAMF treatment durations (p <0.0001 for all three dosing durations). These results demonstrate that biofilm can be effectively eliminated (i.e., significantly reduced) by combined treatment of imaf and ciprofloxacin for multiple dosing durations. Indeed, only 3 iAMF exposures in 15 minutes together with ciprofloxacin are sufficient to effectively eliminate (i.e., significantly reduce) the pseudomonas aeruginosa biofilm.

A similar pattern was observed for the iAMF and antibiotic treatments of staphylococcus aureus biofilms. In addition to being a gram-positive pathogen with several structural and metabolic differences compared to pseudomonas aeruginosa, staphylococcus aureus is of clinical importance as one of the more common pathogens associated with metal implant infections. Staphylococcus aureus (UAMS 1) biofilms were treated with iAMF and antibiotics, alone and in combination. Two antibiotics commonly used in clinic were selected: ceftriaxone (2. Mu.g/mL) and linezolid (2. Mu.g/mL). These concentrations represent the Minimum Inhibitory Concentration (MIC) of the strain. As in previous experiments, the iAMF dose was delivered at 0 and 12 hours. Each dose consisted of the iAMF exposure with the following specifications: tmax=65 ℃, Δtexp=5 minutes, tdose=15 minutes. For treatment with imaf and 2 μg/mL ceftriaxone (fig. 5A), biofilm CFU was initially reduced by more than 3 log, suggesting that staphylococcus aureus biofilm had greater sensitivity (3.29 log reduction) to imaf dosing alone compared to pseudomonas aeruginosa with the same 15 minute dose of imaf (0.96 log reduction). As observed with PA01, between doses, biofilm CFU returned to control levels for the iAMF-only group. Incubation with cef Qu Songwen alone resulted in only about a 2 log reduction after 24 hours. However, CFU decrease after 24 hours was significantly greater (p < 0.0001) when treated in combination with iAMF, where CFU was near the detection limit. At 24 hours, the iAMF and ceftriaxone (2 μg/rnL) or the iAMF and linezolid (2 μg/rnL) showed significantly lower CFU than the antibiotic alone (fig. 5B; p=0.0004 for ceftriaxone and p <0.0001 for linezolid).

The life of a biofilm may vary in actual clinical situations. Applicant investigated whether the combination of iAMF and antibiotic could eliminate (i.e. significantly reduce) more mature biofilms over 48 hours (2 days). Pseudomonas aeruginosa (PAO 1) and Staphylococcus aureus (UAMS 1) biofilms were cultured for 7 days and the same experimental conditions were performed using iAMF at Tmax=65℃withrespect to the biofilms of 2 days. A similar decrease in CFU as the 2 day biofilm was observed. CFU changes followed the same trend as previously observed when treated with the same iAMF dose (tmax=65 ℃, Δtexp=5 min, tdose=15 min) and antibiotic (0.5 μg ml-1 ciprofloxacin for PAO1 and 2 μg ml-1 linezolid for UAMS 1) used with 2 day biofilm (fig. 20A, 20B). There was no significant difference in the magnitude of biofilm reduction for iAMF and antibiotics for 2 and 7 days of biofilm (fig. 20C, 20D).

Antibiotic resistance is becoming more and more common. The treatment of biofilm-associated implant infections is only further complicated by multi-drug resistant pathogens (MDR). The mechanism of the synergistic reaction between antibiotics and iAMF remains unknown. The applicant has claimed that one possible mechanism may be associated with thermally induced membrane rupture, allowing for increased antibiotic absorption. To test whether iAMF can enhance antibiotic activity in MDR pathogens and depending on the resistance mechanism, applicant utilized MDR pseudomonas aeruginosa isolate (MB 699) characterized both genomically and phenotypically. Genomic sequencing was performed on the clinical isolate as previously described (reference 34). It is an MDR isolate with a Minimum Inhibitory Concentration (MIC) of 64 μg/mL for both ciprofloxacin and meropenem. Genomic analysis revealed mutations in DNA gyrase (gyrA, p.thr83ile) and topoisomerase IV (parC, p.ser87leu), which are associated with ciprofloxacin resistance, porin (oprD, p.thr103ser, p.lys115thr, p.phe170leu, p.glu185gin, p.pro186glyfs 35, p.thr187profs 52, p.val189del, p.gly425 ala), which are associated with meropenem resistance. It is assumed that iAMF will enhance the activity of meropenem but not ciprofloxacin. MB699 biofilm was treated with iAMF using the following parameters: tmax=65 degrees celsius, Δtexp=5 minutes, nexp=12, ndose=2, Δtdose=24 hours. Antibiotic administration followed the same protocol as for the PAO1 experiment, and each antibiotic was administered at its lowest inhibitory concentration. (FIG. 11). After 2 doses (0 and 24 hours) and CFU measured at 48 hours, bacterial load was near the detection limit of treatment with imaf and meropenem, whereas ciprofloxacin and imaf did not result in further decrease of CFU compared to imaf or antibiotic alone (fig. 6A). A decrease in meropenem along with iAMF was also observed at sub-MIC concentrations (32. Mu.g/mL) (p <0.0001; FIG. 6B). When combined with imaf, increasing the concentration of ciprofloxacin gradually did not result in an enhanced decrease in CFU. The effect of iAMF and meropenem on MB699 compared to ciprofloxacin was observed by Scanning Electron Microscopy (SEM). The biofilm was fixed and imaged as described when MB699 biofilm was post-treated with imaf (tmax=65 degrees celsius, Δtexp=5 minutes, nexp=12) for 12 hours and incubated continuously with 64 μg/mL ciprofloxacin or meropenem. No obvious morphological changes were observed in the bacteria for treatments with ciprofloxacin, meropenem or iAMF alone. With iAMF and ciprofloxacin, some changes were observed, with slight bacterial elongation and increased membrane wrinkling. Treatment with imaf and meropenem showed fragmented and deformed bacterial cells (fig. 8).

Evidence A discussion

Although the effect of heat on bacterial killing has been known for many years, there are major obstacles to utilizing heat for antibacterial effects in humans. Studies conducted by applicant and others have shown a strong therapeutic effect of heat and antibiotics produced via AMF on eradication (i.e., significant reduction) of biofilm (references 20, 23, 24). Previous studies by our study group demonstrated that after AMF treatment, pseudomonas aeruginosa biofilms were more susceptible to ciprofloxacin (reference 22). Pijls et al (references 24, 35) reported similar results as seen in the study, namely the presence of enhanced effects in staphylococcus epidermidis (Staphylococcus epidermidis) and staphylococcus aureus biofilms on titanium alloys using AMF and antibiotics, than with either treatment alone. One concern of clinical adoption of AMF relates to the therapeutic index, particularly the ability to reduce biofilm by thermal effects while minimizing adjacent tissue damage. Applicants have developed a method, intermittent AMF, that can deliver AMF to an infected metal implant, which can help to address these goals of maintaining efficacy while limiting any toxicity. The precondition for the iAMF is that a brief exposure to the surface of the implant with sufficient cooling time between exposures will result in a therapeutic dose that is capable of eradicating (i.e., significantly reducing) the biofilm while protecting surrounding tissue from injury.

Applicant has demonstrated that even several seconds of iAMF exposure can reduce biofilm load by 1-2 log. However, without more frequent dosing, regrowth returned to baseline within 12 hours. While more frequent administration of imaf may be used, alternative methods used by embodiments include use of imaf to enhance the activity of antibiotics. As has been reported previously, the antibiotics used in the study are not affected by the heat generated by the iAMF and remain stable at these temperatures (references 36, 37). The combined iAMF and antibiotic resulted in a significant reduction in biofilm burden compared to either treatment alone. Importantly, the effect is not limited to one pathogen or one antibiotic. Applicant has demonstrated that both clinically important gram positive (staphylococcus aureus) and gram negative pathogens (pseudomonas aeruginosa), as well as the activity of various antibiotics, are enhanced by iAMF. Since diseases such as PJI are caused by a number of different bacterial pathogens, one goal of developing imasf is to have a treatment that is effective regardless of the pathogen found. Applicants have also demonstrated that the combination of imaf and antibiotic can effectively eliminate (i.e., significantly reduce) biofilm of different lifetimes. Importantly, the effect of the treatment is not seen on the plastic ring, thus indicating the principle underlying the formation of an electrical current between the AMF and the metal. In addition to quantitatively reducing bacterial load, microscopy qualitatively supported the enhanced effects of iAMF and antibiotics.

Biofilms resist antibiotic therapy for a variety of reasons. This includes the difficulty in obtaining sufficient concentrations of drug for targets (bacteria) embedded within the biofilm matrix, and the difficulty for immune cells to reach these pathogens. This creates an environment where biofilm-associated pathogens may be functionally antibiotic resistant. The increasing rate of antibiotic resistance seen worldwide will only further complicate the treatment of biofilm-related infections. One of the most significant findings we have studied is the ability to reduce certain multi-drug resistant bacteria based on resistance mechanisms. Applicants utilized the genome and phenotypically characterized pseudomonas strains to begin understanding the mechanism of action explaining the synergy of iAMF with antibiotics. Applicants claim that the iAMF breaks the bacterial membrane and if the mechanism of resistance is membrane-based (i.e., porin or efflux mechanism), embodiments can reduce MDR strains with antibiotics. However, applicants claim that the chromosome-based resistance mechanism (i.e., gyrase mutation) will not be affected by the combination of the iAMF and the antibiotic, as compared to either alone. Embodiments support these arguments. Applicants were able to show synergy with the use of imaf and meropenem in the MDR strain with known mutations in porin oprD, but not with ciprofloxacin, since the strain contains DNA gyrase mutations. The data support membrane rupture is likely to be an important component, although there are other potential mechanisms that can explain the interaction between the iAMF and the antibiotic in the biofilm context.

Applicant uses iAMF and antibiotics in vitro to effectively eliminate (i.e., significantly reduce) biofilm on metal implants. The water bath experiment in combination with defining the heat exposure time as the "dose" of antimicrobial agent does indeed support the belief that a synergistic interaction between the iAMF and the antibiotic is being seen.

Embodiments help to discuss many previously unknown factors about the final adoption of the iAMF in a clinical setting. This includes the optimal number of iAMF doses that will result in a sustained therapeutic response, as well as the optimal target temperature that will maintain efficacy while minimizing any potential safety concerns. The various embodiments described herein provide valid ranges for these values. Nonetheless, future and ongoing research includes exploring the safety and efficacy of iAMF in large animal models of implant infection. Furthermore, other possible mechanisms of the interaction remain to be explored.

Evidence a materials and methods

In vitro AMF system

A custom design system consisting of multiple solenoid coils was constructed to provide programmed AMF exposure to a stainless steel ring with existing biofilm held in a 50mL conical tube. The parameters of the AMF exposure are specified using custom developed software running on a personal computer. A function generator (33250A,Agilent Technologies) is used to generate the RF signal. The signal is input into a 1000WRF amplifier (1140 la, electronics & innovation) and the amplified signal is directed to the appropriate coil using a USB-controlled relay system. Each coil was constructed using a 0.25 inch diameter copper tube forming a 6 turn solenoid with a 1cm turn-to-turn spacing (fig. 1A). The coil diameter was chosen to accommodate a 50mL conical tube holding the infected loop and culture medium. A plastic bracket is included in each cone to hold the ring in place, thus maintaining the orientation through all coils. The coil is electrically driven as a parallel resonant circuit using a capacitor selected to tune the resonant frequency to about 500 kHz. The operating frequency of the coil is in the range 507 to 522kHz. A matching inductor (matching inductor) is also included in series with the resonant circuit to transform the impedance of each coil to 50 ohms for efficient power transfer. The complete system includes four insulated boxes, each containing eight coils, enabling up to 32 samples to be processed in a single experiment using the iAMF. For the experiments described herein, the coil was operated at 8Vpp with a 50% duty cycle (100 ms/200 ms). A circulation fan (Miller Manufacturing, MN, usa) with an integrated heater was also mounted in each cartridge to maintain the samples at 37 ℃ during the extended length of the experiment.

Characterization and calibration. The strength of the alternating magnetic field in the coil was characterized using a commercial 2D magnetic field probe (AMF life systems, inc., MI, usa). A current probe (TRCP 3000 Rogowski current probes, tektronix inc., OR, usa) was also used to measure the current through the coil during operation.

To characterize AMF heating, the uninfected metal ring was exposed for various durations to reach the desired maximum temperature. The temperature of each ring exposed to the AMF was measured using a fiber optic temperature sensor (PRB-G40-2M-STM-MRI, osensa Innovations, burnaby, BC, canada) attached to the center of the inner surface of the ring with high-temperature epoxy (Epotek 353ND,Epoxy Technologies,CA, usa). Tests were performed to confirm that the epoxy was not affected by the AMF and that no false heating occurred. The ring temperature was recorded using a laptop at a frequency of 2 Hz. The use of fiber optic temperature sensors enables accurate temperature characterization during AMF exposure because they are immune to electromagnetic interference.

Finite element analysis simulation a finite element simulation was performed using commercial simulation software COMSOL Multiphysics (Comsol v5.5, los Angeles, CA, usa) to model the interaction between AMF and metal implants and to investigate the uniformity and magnitude of AMF-induced heating. Quasi-static approximations of maxwell's equations and Penne's biological heat transfer model are used for electromagnetic and thermal simulations. The thermal dose is calculated as cumulative equivalent minutes (CEM 43) (reference 38), which gives the time-temperature relationship in equivalent minutes as follows

Where R is the temperature dependence of the cell death rate (r=0.5 for T >43, r=0.25 for 43+.t+.39), dt is the time interval, to and tfinal are the initial and final heating periods in minutes, respectively. The thermal toxicity due to implant heating was determined based on tissue injury radius CEM240 minutes (irreversible injury) from the implant surface (references 27, 28).

FIG. 1A shows a 3D physical model for simulating metal loops in aqueous biological media in a coil. The coil geometry and current measured in the previous section were used for 3D modeling and initial conditions of 37 degrees celsius were selected for simulation. The physical properties used for the simulation are listed in tables S1, 26. Simulations were performed using free tetrahedral meshing with boundary layers (free tetrahedral meshing). A grid independence study (Grid independent studies) was performed from thicker to thinner grids, determining the optimal number of 186,634 elements to be used for analysis.

In vitro AMF treatment

The iAMF process parameters. The structure and timing control of the iAMF process is shown in fig. 1B. The treatment was organized as a series of doses (Ndose), each dose being spaced apart by a fixed time (atdose). The duration of the iAMF dose ranges from 15 minutes to several hours. Ndose is the number of doses in the overall treatment. Each dose consisted of multiple AMF exposures. During each exposure, the AMF was actuated for a few seconds and the ring was heated. The exposures are separated by a fixed time interval (Δtexp) to allow the ring to cool to the original temperature between the exposures. (Nexp) is the number of exposures performed in one dose of iAMF. Typical exposed heating with a specified target temperature, tmax, and cooling back to baseline temperature in 3-5 minutes is shown. Temperature profiles of three different Tmax values (50, 65 and 80) are also shown. The target temperature is achieved by varying the duration of AMF exposure in the coil. For the iAMF treatment at tmax=80℃, the temperature reached 80 ℃ in 6 seconds and was maintained for up to 12 seconds during the initial construction of the system. Thus, the iAMF heating mode was used for the following tmax=80 ℃ iAMF experiment.

Biofilms were grown using the gram negative pathogen Pseudomonas aeruginosa (PAO 1: ATCC strain: PAO1-GFP: supplied by Joanna Goldberg, MB699: supplied by Sam Shelburn) or the gram positive pathogen Staphylococcus aureus (UAMS 1, supplied by M.Smeltzer) on stainless steel rings (316L, 3/4 "OD, 0.035" wall thickness, 0.2 "height, cut by McMaster Carr, P/N89785K857, U.S.) or titanium rings (5 grade, 3/4" OD,0.035 "wall thickness, 0.2" height, cut by McMaster Carr, P/N89835K 93, U.S.). For Pseudomonas aeruginosa biofilms, isolated colonies were inoculated into 3mL of cation conditioned Mueller Hinton II (MHII) medium (Becton-Dickinson, thermo-Fisher Scientific) and incubated at 220RPM for 18 hours at 37 ℃. The working solution was prepared by adding the culture to sterile Phosphate Buffered Saline (PBS). Bacterial concentration was adjusted with MHII at 600nm using a UV spectrophotometer (Genesys 20,Thermal Scientific) until the Optical Density (OD) reading was between 0.07 and 0.08, indicating a concentration of 108CFU mL-1. The working solution was then diluted to obtain a bacterial concentration of 5X 105CFU mL-1. Biofilms were prepared on each metal ring by placing the ring into 5mL of bacterial solution in a 50mL conical tube. The immersed ring was then incubated in a shaking incubator (Innova 42, new Brunswick Scientific) at 110RPM for 48 hours at 37 ℃. Medium was supplemented halfway at 24 hours by replacing the solution with 5mL fresh MHII. Biofilms prepared with staphylococcus aureus follow the same protocol using trypticase soy broth (TSB, becton-Dickinson of Thermo-Fisher Scientific). Biofilms other than those of the study for 7 days were prepared using the protocol. For biofilms of 7 days old, the loops were similarly cultured, but the culture time was prolonged to 7 days with medium supplementation every 24 hours.

Biofilm preparation, treatment and quantification. The multi-coil system described above was used to study the response of biofilm (pseudomonas aeruginosa or staphylococcus aureus) grown on stainless steel rings to AMF. The biofilm-coated loops were transferred to 50mL conical tubes each with 10mL fresh medium containing the antibiotic at the set concentration. The tubes of fresh medium were preheated to 37 ℃ in a multi-coil system prior to transfer. After transferring the ring to the tube, a sterile 3D-printed ring holder is placed on top of the ring to maintain its orientation in the coil during AMF exposure. The ring is then exposed to intermittent AMF according to the treatment protocol. After each intermittent dose, the ring was rinsed in 10mL of fresh antibiotic-containing medium to remove planktonic bacteria. The loop was then transferred again to 10mL of fresh antibiotic-containing medium and incubated at 37 degrees celsius. After a fixed period of time (typically 12-24 hours), the ring is exposed to a second dose of AMF using the same protocol, and the ring is again incubated in 10mL of medium containing the antibiotic for a further 12-24 hours at 37 degrees celsius. Before and after each dose of iAMF, and at the treatment endpoint, the rings were harvested and rinsed in 5mL PBS and then transferred to 4mL PBS. The rings were sonicated in an ultrasonic water bath for 5 minutes and bacterial density on the ring surface was quantified by plating onto blood agar plates (TSA w/sheep blood, thermo Fisher Scientific) using standard serial dilution drop methods. Three biological replicates were obtained for each experimental condition, and each experiment was repeated using three techniques. All studied control groups included rings that were not exposed to antibiotics or AMF, and rings that were exposed to iAMF or antibiotics as monotherapy. All control groups underwent multiple rinsing and transfer steps to calculate any bacterial losses. A two-factor anova model was used to compare bacterial loads at different time points for single or combination treatments.

The final control group involved the iAMF treatment of infected plastic rings with the same dimensions as the metal rings to confirm that the observed effect was caused by the interaction between AMF and metal. See fig. 7-11 for further details.

Experiments with different AMF target temperatures (Tmax) delivered three unique iAMF treatment algorithms to loops infected with PAO1 biofilm. For all treatments, the rings were incubated with ciprofloxacin (0.5 μg/mL) in 10 mM HII medium at 37 degrees Celsius. Each treatment reached a different target temperature and had a different number of exposures per dose, as depicted in fig. 10. The doses were repeated at 0 and 12 hours.

Although the multiple parameters in each setting are different, the goal is to balance the maximum temperature with the number of exposures to maintain a safe level. These choices depend on the biological heat transfer simulation that we are conducting (not shown). Based on the simulation, each of these AMF treatment combinations was predicted to be safe in terms of tissue damage surrounding the implant.

Experiments with variable AMF dose duration in combination with antibiotic treatment biofilms of pseudomonas aeruginosa strain PAO1 were prepared on stainless steel rings using the same culture protocol as described above and incubated with 0.5 μg/mL ciprofloxacin in 10mL MHII medium at 37 degrees celsius. The ring was exposed to imaf to Tmax at 65 degrees celsius with a 5 minute exposure interval. The duration of each dose of iAMF ranged from 15 minutes to 1 hour (3 to 12 exposures). Doses were delivered at 0 and 12 hours and the cyclic biofilm load was quantified at various time points as described above. For staphylococcus aureus experiments, a biofilm of UAMS1 was prepared on stainless steel rings according to the culture protocol and incubated with 2 μg/mL ceftriaxone or 2 μg/mL linezolid in 10mL TSB medium. The ring was exposed to imaf to Tmax at 65 degrees celsius, with 5 minutes between each exposure, with each dose for a duration of 15 minutes (3 exposures). Doses were delivered at 0 and 12 hours and biofilm loading was quantified at 24 hours.

Treatment with combined iAMF and antibiotic A biofilm of MB699 (MDR-strain of Pseudomonas aeruginosa) was incubated with ciprofloxacin (64 or 128 μg/mL) or meropenem (32 or 64 μg/mL) in 10mL MHII medium. The ring was exposed to imaf to Tmax at 65 degrees celsius for 5 minutes with each dose for a duration of 1 hour. Doses were delivered at 0 and 24 hours, and loop biofilm loading was quantified at 48 hours.

Imaging system

Laser scanning confocal microscopy biofilms cultured from p ao1 pseudomonas aeruginosa (GFP-PAO 1) expressing Green Fluorescent Protein (GFP) were prepared on loops using the procedure described above, then exposed to isamf (tmax=65 degrees celsius, Δtexp=5 minutes, dosing duration 1 hour) and incubated with 0.5 μg/mL ciprofloxacin in 10mL MHII medium for 12 hours. After rinsing in 5mL DPBS, the ring was then fixed in 5% glutaraldehyde (Sigma Aldrich, st.louis, MO) for 30 min at 37 ℃ and protected from light. The loop was then rinsed in 5mL DPBS to remove excess glutaraldehyde and incubated in 200 μg/mL concanavalin A-Alexa Fluor 647 conjugate (Life Technologies, grand Island, NY) for 15 minutes in the dark at room temperature to stain EPS. After staining, the rings were mounted on 50mm glass bottom plates and images were captured using a Zeiss LSM880 Airyscan laser confocal microscope. GFP-PA01 bacteria and ConA-stained EPS were imaged using a 40X objective. Multiple regions of the ring surface were randomly selected and Z-stacks (stacks) were acquired with a slice step size of 0.5 μm. Prior to image processing, the Z-stack was deconvoluted using autopant x 3 (Media Cybernetics, MD, usa) to improve X, Y and Z-direction image resolution. Deconvoluted images were analyzed using Imaris x649.1.2 (Bitplane AG, zurich, switzerland).

Scanning Electron Microscopy (SEM) biofilms cultured from pseudomonas aeruginosa (MB 699) were prepared on rings and exposed to isamf (tmax=65 degrees celsius, aterp=5 minutes, administration duration 1 hour) and incubated with 64 μg/mL ciprofloxacin or 64 μg/mL meropenem for 12 hours in 10mL MHII medium. Then, a ring with biofilm was prepared for SEM following a similar procedure as described previously (reference 40). The ring was carefully transferred to 4mL pbs, rinsed 3 times in 4mL0.1M sodium dimethylarsinate buffer, and fixed in 4mL 2% glutaraldehyde, 2% paraformaldehyde in 0.1M sodium dimethylarsinate buffer for 24 hours. After 3 washes in 4mL of sodium dimethylarsinate buffer, the samples were re-fixed (re-fix) in 4mL of 2% osmium in 0.1M sodium dimethylarsinate buffer for 2 hours. The ring was then rinsed five more times with 4mL of deionized water and dehydrated in five steps at room temperature by placing the ring into 4mL of 50, 70 (twice), 85, 95 (twice) and 100% ethanol for 5 minutes each. The ring was then transferred successively to 4mL of 25, 50, 75 and 100% (twice) Hexamethyldisilazane (HMDS) in ethanol for 15 minutes each. Finally, the samples were dried in a fume hood for 24 hours. Samples were mounted on aluminum stubs, gold/palladium sputter coated, and examined using a Zeiss SigmaVP scanning electron microscope. The image was obtained at 10kV with a magnification of approximately 35000X.

And (5) statistics. Significance was determined by two-factor anova followed by Tukey's multiple comparison test as described for the in vitro AMF treatment. "n" represents the number of biological replicates. Each biological repeat experiment was performed 2 or 3 technical repeat experiments. All analyses were performed using GraphPad Prism version 8.4.3 (San Diego, CA), and p-values <0.05 were considered statistically significant.

Evidence A supplementary Material

Epoxy resistance to imaf (Epotek 353 ND) the fiber optic thermal sensor was glued to Epotek 353ND epoxy at the tip and placed in a 10mL DPBS. Bare sensors are also placed in the DPBS. The distance between the tips of the two sensors was 1cm. The iAMF (tmax=65℃) was applied for 10 minutes and the temperature readings of the two sensors were recorded and compared.

Determination of synergy between heat and antibiotics in biological films. The synergy of heat and ciprofloxacin in the biofilm was determined using a partial inhibitory concentration (FIC) index (supplementary references 1-3). The FIC index was calculated based on the Minimum Biofilm Eradication Concentration (MBEC), defined as the minimum concentration of antimicrobial material that eradicates 99.9% of the biofilm embedded bacteria (3 log reduction in CFU mL-1) compared to the growth control. The heat treatment time was regarded as antimicrobial substance dose, and heat-treated MBEC was defined as the shortest treatment time to eliminate 99.9% of biofilm-embedded bacteria (supplementary reference 4). Thus, the equation for FIC index calculation with heat treatment and antibiotics can be derived: fic= (CHeat/mbechoat) + (CAbx/mbechabx), where mbechoat and mbechabx are MBECs of heat treatment and antibiotic concentration, respectively, alone, and CHeat and CAbx are heat treatment time and antibiotic concentration, respectively, in combination. FIC values of ∈0.5 are considered synergistic, values >0.5 and <4 indicate no interaction or additivity, and values greater than or equal to 4 indicate antagonism (supplementary references 3, 4).

A temperature controlled water bath (model 1235,VWR Scientific) was used for heat treatment. A50 mL tube with 10mL fresh MHII was placed in a water bath and preheated to 65℃containing a concentration of ciprofloxacin. PAO1 biofilms were prepared as described above. The PAO1 biofilm coated rings were transferred to a pre-heated 50mL conical tube and exposed to heated medium for target duration. Immediately after heat exposure, the ring with biofilm was transferred to a 50mL conical tube at 37 ℃ to set a concentration of 10mL fresh medium with ciprofloxacin. The ring was then incubated at 37 ℃. After 12 hours or 24 hours, the rings were harvested and rinsed in 5mL sterile PBS and then transferred to 4mL PBS. After 5 minutes of sonication in an ultrasonic bath, the bacterial density on the loop was counted using a standard continuous plating method to determine cfum-2.

Further discussion of embodiments

AMF is a non-invasive method of treating implant-related infections in which external transducer coils produce time-varying AMF in the vicinity of a metallic implant in the body. AMF produces surface currents on the implant, which can eradicate (i.e., significantly reduce) the pathogen. In the case of an infected implant, bacteria, possibly in the form of biofilms, adhere to the surface. The localized currents may be used to eradicate (i.e., significantly reduce) the pathogen or sensitize it to antimicrobial therapy.

Embodiments relate to inducing very high currents for very short periods of time, resulting in little or no heating of surrounding tissue, but with an antibacterial effect similar to previous treatment methods resulting in higher tissue temperatures. Thus, embodiments use the lower temperature and antibacterial effect of AMF to address the problem (tissue damage due to heat when attempting to treat a biofilm) to reduce the risk of thermal damage to surrounding tissue.

Embodiments demonstrate that the duty cycle of AMF exposure has an effect on temperature rise. When the metal ring is exposed to AMF, an exposure of 1ms duration (0.1% duty cycle) with a period of 1 second results in a total temperature rise of less than 5-6 degrees celsius in 2 hours. Similar heating was observed for 0.1% duty cycle exposure (10 ms every 10 seconds, 40ms every 40 seconds) for different durations. Embodiments show a 40ms decrease in CFU exposure in the presence of antibiotics.

When used with antibiotics, pulsed exposure has an increased effect.

Exposure using a brief pulse can produce high currents on the implant without significant temperature rise. This enhances the safety of the embodiments compared to using exposures designed to reach the treatment temperature (60-80 degrees celsius). However, in the case of some embodiments, longer exposure and brief exposure are required to achieve a therapeutic effect. Further, the effectiveness of some embodiments depends on the concentration of the antibiotic administered. In some embodiments, a mixing method is used (which uses a temperature sufficient to produce an inflammatory response that in turn triggers the immune system). The temperature rise can be controlled by varying the duty cycle of the process.

While the previous disclosure discusses the use of heat as a direct antibacterial, the mechanism of action of the low temperature embodiment may include: (a) mechanical disruption of the biofilm matrix (which allows for better penetration of the antibiotic and the ability of the antibiotic to reach its target), (b) stimulation of otherwise 'dormant' metabolically inactive organisms which are now susceptible to specific antimicrobial agents, (c) or combinations of the above.

As seen with high temperature AMF, low temperature AMF may be synergistic with multiple antimicrobial agents and may not be limited to a single chemical class of drug. Thus, embodiments may be broadly applicable to bacterial and fungal infections or any pathogen that can form a biofilm on a metal implant.

Fig. 14 includes a block diagram of an example system in which embodiments may be used. As seen, the system 900 may be a smartphone or other wireless communication device or any other internet of things (Internet of Things) (IoT) device. The baseband processor 905 is configured to perform various signal processing with respect to communication signals to be transmitted from or received by the system. The baseband processor 905 is in turn coupled to an application processor 910, which may be the main CPU of the system to execute the OS and other system software in addition to user applications such as many well-known social media and multimedia applications. The application processor 910 may be further configured to perform a variety of other computing operations for the device.

The application processor 910, in turn, may be coupled to a user interface/display 920 (e.g., a touch screen display). In addition, the application processor 910 may be coupled to a memory system that includes non-volatile memory (i.e., flash memory 930) and system memory (i.e., DRAM 935). As further seen, the application processor 910 is also coupled to a capture device 945, such as one or more image capture devices that may record video and/or still images.

A Universal Integrated Circuit Card (UICC) 940 includes a subscriber identity module (subscriber identity module) that in some embodiments includes secure memory to store secure user information. The system 900 may further include a security processor 950 (e.g., a trusted platform module (Trusted Platform Module) (TPM)) that may be coupled to the application processor 910. A plurality of sensors 925, including one or more multi-axis accelerometers (multi-axis accelerometers), may be coupled to the application processor 910 to enable input of a variety of sensed information, such as motion and other environmental information. In addition, one or more authentication devices (authentication devices) may be used to receive, for example, user biometric input for use in an authentication operation.

As further illustrated, a near field communication (near field communication) (NFC) contactless interface 960 is provided that communicates in the NFC near field via an NFC antenna 965. Although a single antenna is shown, it should be appreciated that in some implementations, one antenna or a different set of antennas may be provided to enable various wireless functionalities.

A power management integrated circuit (power management integrated circuit) (PMIC) 915 is coupled to the application processor 910 to perform platform level power management. To this end, the PMIC 915 may issue power management requests to the application processor 910 to enter certain low power states as desired. Additionally, the PMIC 915 may also control power levels of other components of the system 900 based on platform constraints.

To enable communication to be sent and received, such as in one or more internet of things (IoT) networks, various circuits may be coupled between baseband processor 905 and antenna 990. In particular, there may be a Radio Frequency (RF) radio transceiver 970 and a Wireless Local Area Network (WLAN) radio transceiver 975. In general, RF radio transceiver 970 may be used to receive and transmit wireless data and calls (calls) according to a given wireless communication protocol, such as a 5G wireless communication protocol, e.g., according to code division multiple access (code division multiple access) (CDMA), global system for mobile communications (global system for mobile communication) (GSM), long term evolution (long term evolution) (LTE), or other protocols. In addition, there may be a GPS sensor 980 with location information provided to the security processor 950. Other wireless communications may also be provided, such as the reception or transmission of radio signals (e.g., AM/FM) and other signals. In addition, local wireless communication, such as in accordance with Bluetooth, may also be implemented via WLAN radio transceiver 975 ^TM Or the IEEE802.11 standard.

Fig. 15 shows a block diagram of a system according to another embodiment of the invention. Multiprocessor system 1000 is a point-to-point interconnect system, such as a server system, and includes a first processor 1070 and a second processor 1080 coupled via a point-to-point interconnect 1050. Each of processors 1070 and 1080 may be multicore processors, such as SoCs, including first and second processor cores (i.e., processor cores 1074a and 1074b and processor cores 1084a and 1084 b), although many more cores may be present in the processors. Further, processors 1070 and 1080 may each include a power controller unit (power controller unit) 1075 and 1085. Further, processors 1070 and 1080 may each include a security engine (security engine) to perform security operations such as attestation, ioT network onboard (network onboarding), and so forth.

The first processor 1070 further includes a memory controller hub (memory controller hub) (MCH) 1072 and point-to-point (P-P) interfaces 1076 and 1078. Similarly, second processor 1080 includes a MCH1082 and P-P interfaces 1086 and 1088.MCH1072 and 1082 couple the processors to respective memories, namely a memory 1032 and a memory 1034, which may be portions of main memory (e.g., DRAM) locally attached to the respective processors. First processor 1070 and second processor 1080 may be coupled to a chipset (chipset) 1090 via P-P interconnects 1062 and 1064, respectively. Chipset 1090 includes P-P interfaces 1094 and 1098.

In addition, chipset 1090 includes an interface 1092 to couple chipset 1090 with a high performance graphics engine (high-performance graphics engine) 1038 via a P-P interconnect 1039. In turn, chipset 1090 may be coupled to a first bus 1016 via an interface 1096. Various input/output (I/O) devices 1014 may be coupled to first bus 1016, along with a bus bridge 1018 that couples first bus 1016 to a second bus 1020. Various devices may be coupled to the second bus 1020 including, for example, a keyboard/mouse 1022, communication devices 1026, and a data storage unit 1028 such as a nonvolatile memory or other mass storage device. As seen, in one embodiment, data storage unit 1028 may include code 1030. As further seen, data storage unit 1028 also includes trusted storage 1029 to store sensitive information to be protected. Further, an audio I/O1024 may be coupled to the second bus 1020.

Fig. 16 depicts an IoT environment that may include a wearable device (small form factor) or other small form factor (IoT) device. In one particular implementation, the wearable module 1300 may beCurie ^TM A module comprising a plurality of components that fit within a single small module, which may be implemented as all or part of a wearable device. As seen, module 1300 includes a core 1310 (of course, in other embodiments more than one core may be present). Such cores may be relatively low complexity ordered (in-order) cores, e.g. based on Intel + >Quark ^TM And (5) designing. In some embodiments, core 1310 may implement a trusted execution environment (Trusted Execution Environment) (TEE). Core 1310 is coupled to various components including a sensor hub 1320, which may be configured to interact with a plurality of sensors 1380, such as one or more biometric, motion, environmental, or other sensors. There is a power delivery circuit (power delivery circuit) 1330 and a non-volatile memory 1340. In embodiments, the circuit may include a rechargeable battery and a recharging circuit, which in one embodiment may receive charging power wirelessly. There may be one or more input/output (IO) interfaces 1350, such as one or more interfaces compatible with one or more of the USB/SPI/I2C/GPIO protocols. Furthermore, there is a wireless radio transceiver 1390, which may be Bluetooth ^TM Low energy or other short range wireless radio transceivers to enable wireless communications as described herein. In different implementations, the wearable module may take many other forms. The wearable and/or IoT device has a small form factor (small form factor), low power requirements, a limited instruction set, relatively slow computational throughput, or any of the above, as compared to a typical general purpose CPU or GPU.

Embodiments may be used in many different types of systems. For example, in one embodiment, a communication device may be arranged to perform the various methods and techniques described herein. Of course, the scope of the invention is not limited to communication devices, and instead other embodiments may involve other types of apparatus for processing instructions, or include one or more machine-readable media that in response to execution of instructions on a computing device cause the device to perform one or more of the methods and techniques described herein.

Program instructions may be used to implement the operations described herein using a general-purpose or special-purpose processing system that is programmed with the instructions. In another aspect, operations may be performed by specific hardware components that contain hardwired logic for performing the operations, or by any combination of programmed computer components and custom hardware components. The methods described herein may be provided as (a) a computer program product that may include one or more machine-readable media having stored thereon instructions that may be used to program a processing system or other electronic device to perform the methods, or (b) at least one storage medium having stored thereon instructions for causing a system to perform the methods. The term "machine-readable medium" or "storage medium" used herein shall include any medium (transient medium) that is capable of storing or encoding a sequence of instructions for execution by the machine and that cause the machine to perform any one of the methods described herein, including signals or non-transient media. The term "machine-readable medium" or "storage medium" shall accordingly include, but not be limited to, memories such as solid-state memories, optical and magnetic disks, read-only memories (ROMs), programmable ROMs (PROMs), erasable PROMs (EPROMs), electrically EPROMs (EEPROMs), magnetic disk drives, floppy disks, compact disk ROMs (CD-ROMs), digital versatile disks (digital versatile disk) (DVDs), flash memory, magneto-optical disks, and more exotic mediums such as machine-accessible biological state preservation or signal preservation memories. The medium may include any mechanism for storing, transmitting, or receiving information in a machine-readable form, and the medium may include media through which program code may pass, such as antennas, optical fibers, communication interfaces, etc. Program code may be transmitted in the form of packets (packets), serial data (serial data), parallel data (parallel data), etc., and may be used in compressed or encrypted format. Moreover, software in one form or another (e.g., procedures, processes, methods, applications, modules, logic, etc.) is often referred to in the art as taking an action or causing a result. This expression is merely for the purpose of describing a shorthand way of saying that execution of the software by a processing system causes the processor to perform an action or produce a result.

A module as used herein refers to any hardware, software, firmware, or combination thereof. Often, the module boundaries illustrated as separate typically vary and may overlap. For example, the first and second modules may share hardware, software, firmware, or a combination thereof, while some independent hardware, software, or firmware may be retained. In one embodiment, the use of the term logic includes hardware, such as transistors, registers, or other hardware, such as programmable logic devices. However, in another embodiment, the logic circuitry also includes software or code integrated with hardware, such as firmware or microcode (micro-code).

Various examples of embodiments are now discussed.

Embodiment 1. A system comprising: at least one Alternating Magnetic Field (AMF) transmitter configured to apply one or more AMF pulses to the metal implant; at least one function generator; at least one processor; and at least one machine readable medium having data stored thereon that, if used by the at least one processor, causes the at least one processor, the at least one function generator, and the at least one transmitter to perform operations comprising delivering a plurality of AMF pulses to the metal implant; wherein each of the plurality of AMF pulses has a duty cycle of less than 1% and a period of 1ms to 60 seconds.

The "duty cycle" or power cycle (power cycle) is the fraction of one "cycle" that the signal or system is in an active state. The duty cycle is typically expressed as a percentage or ratio. The period is the time it takes for the signal to complete the on and off periods. The duty cycle (ratio) can be expressed as: d= (PW)/T, where D is the duty cycle, PW is the pulse width (pulse active time), and T is the total period of the signal. Thus, a duty cycle of 60% means that the signal is on 60% of the time, but off 40% of the time. The "on time" of a 60% duty cycle may be a fraction of a second, a day or even a week depending on the length of the period.

In other embodiments, each of the plurality of AMF pulses has a duty cycle of less than 1, 2, 3, 4, 5, or 6%. In other embodiments, each of the plurality of AMF pulses has a period of 0.5 milliseconds to 20 seconds.

A system, comprising: at least one Alternating Magnetic Field (AMF) transmitter configured to apply one or more AMF pulses to the metal implant; at least one function generator; at least one processor; and at least one machine readable medium having data stored thereon that, if used by the at least one processor, causes the at least one processor, the at least one function generator, and the at least one transmitter to perform operations comprising delivering a plurality of AMF pulses to the metal implant; wherein each of the plurality of AMF pulses has a duty cycle of less than 1% and a period of 200ms to 60 seconds.

Embodiment 2. The system of embodiment 1 wherein the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT).

Embodiment 3. The system of any of embodiments 1-2, wherein each of the plurality of pulses has a pulse width of 2ms to 50 ms.

Embodiment 4. The system of any of embodiments 1-3, wherein the operation comprises delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

Embodiment 5. The system of any of embodiments 1-4, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first physical profile and the second metal implant has a second physical profile that is different from the first physical profile; the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

The system of embodiment 4, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

The system of embodiment 4, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; the first protocol includes a first amount of a therapeutic property and the second protocol includes a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

For example, the software may specify, via a user interface, that a user use different processing protocols for different devices. Two different protocols can be used for two different sizes of the same knee implant (knee implant). Two different protocols may be used for two different brands of the same knee implant (one device from manufacturer 1 and the other from manufacturer 2).

Embodiment 5.1. The system of embodiment 5, wherein the physical property comprises one of density (kg/m 3), electrical conductivity (S/m), relative permittivity, or thermal conductivity (W/(m.K)), specific heat (J/(kg.K)).

Embodiment 5.2. The system of any of embodiments 1-5.1 wherein the therapeutic property comprises a total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Fixed time interval (minutes) between two doses (Δt _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) One of them.

Embodiments are manifold and include various ranges and combinations of ranges, such as those found in the tables below. In other words, different frequencies within the ranges in the following table may combine the various exposure durations (or other parameters) within the ranges in the table of fig. 17.

In FIG. 12, N _dose =2 (including dose 1710 and dose 1711). Δt (delta t) _dose Indicated at 1712. t is t _exp Indicated at 1731, 1732, 1733, 1734, 1735, 1736, 1737, 1738. In one embodiment, these values are equal to each other, but inIn other embodiments, these values are not all equal to each other. N (N) _exp =4 and includes exposures 1701, 1702, 1703, 1704 of dose 1710 and exposures 1705, 1706, 1707, 1708 of dose 1711. N (N) _exp Is the same for each of the doses 1710, 1711, but may vary between doses in other embodiments. Δt (delta t) _exp Indicated at 1721, 1722, 1723, 1725, 1726, 1727. In one embodiment, these values are equal to each other, but in other embodiments, these values are not all equal to each other.

In FIG. 12, an example period includes t _exp +Δt _exp . The duty cycle can be expressed as: d= (PW)/T, which in this case includes d= (T) _exp )/(t _exp +Δt _exp )。

Fig. 13 discusses a method 200 that may be performed by at least one processor. As discussed above, various procedures may be determined. For example, a first protocol including a certain duty cycle or other therapeutic characteristic may be designed for a metal stent of a first manufacturer, and a second protocol including a certain duty cycle or other therapeutic characteristic may be designed for a metal stent of a second manufacturer. For example, a first protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a first antibiotic, and a second protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a second antibiotic. For example, a first protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a first dose of a first antibiotic, and a second protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a second dose of the first antibiotic. For example, a first protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a first material (e.g., silver nanoparticles) coating the implant, and a second protocol including a certain duty cycle or other therapeutic characteristic may be designed for use with a second material coating the implant. These protocols may be based on simulations, like those discussed in evidence a. As shown in block 201, the protocol may differ somewhat in pulse width, duty cycle, dose duration, etc.

In block 202, various procedures may be stored in a database, such as the memory discussed in fig. 14, 15, or 16.

In block 203, the user may select a procedure based on his or her knowledge of the implant to be treated. The procedure may also be selected based on other patient specific details, such as the age or weight of the patient, the type of biofilm (e.g., what type of bacteria caused the biofilm), where the implant is located in the patient, etc. However, in block 204, the information may be entered from, for example, a medical record, such that entering medical implant information results in automatic selection of a procedure corresponding to the implant. In block 205, imaging may be used to identify an implant, and upon identification, a protocol specific to the implant may be proposed. The image authentication may be compared to information stored in the medical record. Based on the comparison, the user can select the appropriate procedure (which can be listed in a list that is a subset of all procedures). The protocol may suggest an acceptable range in which the user may select a parameter (e.g., a maximum temperature of 60 to 70 degrees celsius, with the user selecting 68 degrees celsius).

Along with procedure confirmation (block 207), patient treatment (block 208), and patient record update (block 209), then the procedure is entered in block 206.

While various embodiments are directed to metal implants, other embodiments may be used with other materials that still provide conductivity for the current induced by the AMF.

Embodiment 5.21 the system of embodiment 5.2 wherein the therapeutic property comprises T _max ＝65℃、Δt _exp =5 min, t _dose =15 min.

Embodiment 5.22 the system of embodiment 5.2 wherein the therapeutic property comprises T _max <Δt at 80℃for 2 to 7 minutes _exp T of 5 to 60 minutes _dose T less than 10 seconds _exp 。

In some variations of example 5.22, t _exp Less than 50ms. In implementationIn some variations of example 5.22, t _exp From 1ms to 50ms.

In some embodiments, these values are critical values that provide for a brief exposure to the implant surface with sufficient cooling time between exposures, resulting in a therapeutic dose that is capable of eradicating (i.e., significantly reducing) the biofilm while protecting surrounding tissue from injury.

Another version of embodiment 5.22 is the system of embodiment 5.2 wherein the therapeutic property comprises a T of 50 to 80 °c _max Δt of 1 to 10 minutes _exp T of 5 to 120 minutes _dose T less than 10 seconds _exp 。

Embodiment 5.23 the system of embodiment 5.2, wherein the therapeutic property comprises T _max ＝65℃、Δt _exp =5 min, N _exp ＝12、N _dose ＝2、Δt _dose T=24 hours and less than 10 seconds _exp 。

Embodiment 5.231 the system of embodiment 5.2, wherein the therapeutic property comprises T _max =55 to 75 ℃, Δt _exp =2 to 7 min, N _exp =5 to 20, n _dose =1 to 5, Δt _dose T=10 to 30 hours, and 2 to 10 seconds _exp 。

Embodiment 5.24 the system of embodiment 5.2, wherein the therapeutic property is configured to rupture a bacterial membrane of a biological membrane included on the metal implant.

The ability to modulate one or more therapeutic properties unexpectedly provides the ability to reduce certain multi-drug resistant bacteria based on resistance mechanisms.

Embodiment 5.25 the system of embodiment 5.2 wherein the therapeutic property comprises T _max ＝65℃、Δt _exp =5 min, N _exp ＝12、N _dose ＝2、Δt _dose T=24 hours and less than 10 seconds _exp 。

Embodiment 5.26 the system of embodiment 5.2 wherein the therapeutic property comprises T _max =45 to 85 ℃.

Example 5.27 according to example 5The system of 2, wherein the therapeutic property comprises Δt of 2 to 10 minutes _exp 。

Embodiment 5.28 the system of embodiment 5.2 wherein the therapeutic property comprises N of 3 to 50 _exp 。

Embodiment 5.29 the system of embodiment 5.2 wherein the therapeutic property comprises N _dose =1 to 7.

Embodiment 5.30 the system of embodiment 5.2 wherein the therapeutic property comprises Δt _dose =10 to 30 hours.

Embodiment 5.31 the system of embodiment 5.2 wherein the therapeutic property comprises t of 1 to 15 seconds _exp 。

Embodiment 5.32 the system of embodiment 5.2 wherein the therapeutic property comprises a frequency of less than 300 kHz. This helps to reduce damage to the tissue surrounding the implant. Other embodiments are 175 to 225kHz or 150 to 25kHz.

The system of embodiment 4, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first physical characteristic and the second metal implant has a second physical characteristic different from the first physical characteristic; the first protocol includes a first amount of a therapeutic property and the second protocol includes a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

In embodiments, the first physical property relates to a first type of biofilm and the second physical property relates to a second type of biofilm. For example, the first and second types of biological membranes may involve first and second types of bacteria that are different from each other. The protocol may require a greater maximum temperature for the first type of bacteria compared to the second type of bacteria.

The system of embodiment 4, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first physical characteristic and the second metal implant has a second physical characteristic different from the first physical characteristic; the first protocol includes a first therapeutic property and the second protocol includes a second therapeutic property different from the first therapeutic property.

For example, pulse width modulation may be used without a maximum temperature to treat one type of bacteria, while a programmed maximum temperature may be used to treat another type of bacteria.

Embodiment 6. The system of any of embodiments 5-5.2 wherein the first procedure comprises a first period and the second procedure comprises a second period different from the first period.

Embodiment 7. The system of any of embodiments 5-6, wherein the first protocol comprises a first pulse width and the second protocol comprises a second pulse width different from the first pulse width.

Embodiment 8. The system of any of embodiments 5-7, wherein: the first procedure includes applying a first duration of a plurality of pulses to a transmitter, and the second procedure includes applying a second duration of the plurality of pulses to the transmitter; the first duration is different from the second duration.

Embodiment 9. The system of any of embodiments 1-8, wherein the operations comprise delivering a plurality of AMF pulses to the metal implant to raise a temperature on a surface of the metal implant by less than 10 degrees celsius in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 10. The system of any of embodiments 1-9, wherein the operations comprise delivering a plurality of AMF pulses to the metal implant to induce a current of 50 to 3000A/cm 2 on a surface of the metal implant in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 11. The system of any of embodiments 1-10, comprising at least one sensor, wherein the operations comprise: sensing a parameter using the at least one sensor; at least one of the duty cycle or period is changed in response to the sensed parameter.

Embodiment 11.1 the system of embodiment 11, wherein the operation includes changing the treatment characteristic in response to the sensed parameter.

Embodiment 12. The system of any of embodiments 11-11.1 wherein the parameter comprises at least one of sound, temperature, resonance, energy, or a combination thereof.

This may include, for example, sound or temperature in the proximate region of the implant. See, for example, systems such as those described in U.S. patent application publication No. 2019/0159725.

Further, sensing may be coordinated with sensing embedded in or coupled to the implant. For example, if the implant itself has a built-in temperature monitor, such monitor communicates wirelessly (e.g., bluetooth (r), etc.) with the system. Thus, the system can sense the temperature in the vicinity of the device and adjust the therapeutic characteristics (e.g., duty cycle) to adjust the temperature to the target temperature, e.g., T _max Or a percentage thereof.

Embodiment 21. A system comprising: at least one Alternating Magnetic Field (AMF) transmitter configured to apply one or more AMF pulses to the metal implant; at least one function generator; at least one processor; and at least one machine readable medium having data stored thereon that, if used by the at least one processor, causes the at least one processor, the at least one function generator, and the at least one transmitter to perform operations comprising delivering a plurality of AMF pulses to the metal implant; wherein the at least one machine readable medium comprises a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; the first protocol includes a first amount of a therapeutic property and the second protocol includes a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

Embodiment 22. The system of embodiment 21, wherein the physical property comprises one of density (kg/m 3), electrical conductivity (S/m), relative permittivity, or thermal conductivity (W/(m.K)), specific heat (J/(kg.K)).

Embodiment 23. The system of any of embodiments 21-22, wherein the therapeutic property comprises a total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Fixed time interval (minutes) between two doses (Δt _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) One of them.

Embodiment 24. The system of any of embodiments 21-23, wherein the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT).

Embodiment 25. The system of any of embodiments 21-24, wherein each of the plurality of pulses has a pulse width of 2ms to 50 ms.

Embodiment 26. The system of any of embodiments 21-25, wherein the operation comprises delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

Embodiment 27. The system of any of embodiments 21-26 wherein the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

Embodiment 28. The system of embodiment 27 wherein the first duty cycle is less than 1%.

Embodiment 29. The system of any of embodiments 21-28, wherein the first protocol comprises a first period and the second protocol comprises a second period different from the first period.

Embodiment 30. The system of embodiment 28, wherein the first period is 1ms to 60 seconds.

Embodiment 31. The system of any of embodiments 21-30, wherein the first protocol comprises a first pulse width and the second protocol comprises a second pulse width different from the first pulse width.

Embodiment 32. The system of any of embodiments 21-31, wherein: the first procedure includes applying a first duration of a plurality of pulses to a transmitter, and the second procedure includes applying a second duration of the plurality of pulses to the transmitter; the first duration is different from the second duration.

Embodiment 33 the system of any of embodiments 21-32, wherein the operations include delivering a plurality of AMF pulses to the metal implant to raise a temperature on a surface of the metal implant by less than 10 degrees celsius in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 34 the system of any of embodiments 21-33, wherein the operations comprise delivering a plurality of AMF pulses to the metal implant to induce a current of 50 to 3000A/cm 2 on a surface of the metal implant in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 35 the system of any of embodiments 21-34, comprising at least one sensor, wherein the operations comprise: sensing a parameter using the at least one sensor; at least one of the duty cycle or period is changed in response to the sensed parameter.

Embodiment 36. The system of embodiment 35, wherein the operation comprises changing the treatment characteristic in response to the sensed parameter.

Embodiment 37 the system of any of embodiments 35-36, wherein the parameter comprises at least one of sound, temperature, resonance, energy, or a combination thereof.

Embodiment 41. The at least one machine-readable medium of any of embodiments 1-37.

For example, embodiments include software that is independent of the AMF transmitter, function generator, computer, etc.

Embodiment 51. A method performed by at least one processor, comprising: delivering a plurality of AMF pulses to the metal implant in response to a user selecting one of the first or second protocols via the user interface; wherein the first protocol is configured for a first metal implant and the second protocol is configured for a second metal implant; wherein the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; wherein the first protocol comprises a first amount of a therapeutic property and the second protocol comprises a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

Embodiment 52. The method of embodiment 51, wherein the physical property comprises one of density (kg/m≡3), electrical conductivity (S/m), relative permittivity, or thermal conductivity (W/(m. K)), specific heat (J/(kg. K)).

Embodiment 53. The method of any of embodiments 51-52, wherein the therapeutic property comprises a total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Fixed time interval (minutes) between two doses (Δt _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) One of them.

Embodiment 54. The method of any of embodiments 51-53, wherein the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT).

Embodiment 55. The method of any of embodiments 51-54, wherein each of the plurality of pulses has a pulse width of 2ms to 50 ms.

Embodiment 56 the method of any of embodiments 51-55, comprising delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

Embodiment 57. The method of any of embodiments 51-56 wherein the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

Embodiment 58. The method of embodiment 57 wherein the first duty cycle is less than 1%.

Embodiment 59. The method of any one of embodiments 51-58 wherein the first procedure comprises a first period and the second procedure comprises a second period different from the first period.

Embodiment 60. The method of embodiment 58 wherein the first period is 1ms to 60 seconds.

Embodiment 61. The method of any of embodiments 51-60, wherein the first protocol comprises a first pulse width and the second protocol comprises a second pulse width different from the first pulse width.

Embodiment 62. The method of any of embodiments 51-61, wherein: the first procedure includes applying a first duration of a plurality of pulses to a transmitter, and the second procedure includes applying a second duration of the plurality of pulses to the transmitter; the first duration is different from the second duration.

Embodiment 63 the method of any of embodiments 51-62, comprising delivering a plurality of AMF pulses to the metal implant to raise a temperature on a surface of the metal implant by less than 10 degrees celsius in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 64 the method of any of embodiments 51-63, comprising delivering a plurality of AMF pulses to the metal implant to induce a current of 50 to 3000A/cm 2 on a surface of the metal implant in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 65. The method of any of embodiments 51-64, comprising: sensing a parameter using at least one sensor; at least one of the duty cycle or period is changed in response to the sensed parameter.

Embodiment 66. The method of embodiment 65, comprising changing the treatment characteristic in response to the sensed parameter.

Embodiment 67. The method of any of embodiments 65-66, wherein the parameter comprises at least one of sound, temperature, resonance, energy, or a combination thereof.

Example 71. A method comprising: using at least one Alternating Magnetic Field (AMF) transmitter, at least one function generator, at least one processor, and at least one machine readable medium having data stored thereon that, if used by the at least one processor, causes the at least one processor, the at least one function generator, and the at least one transmitter to perform operations comprising delivering a plurality of AMF pulses to the metal implant to deliver a plurality of AMF pulses to the metal implant; wherein each of the plurality of AMF pulses has a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 72. The method of embodiment 71 wherein the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT).

Embodiment 73. The method of any of embodiments 71-7 wherein each of the plurality of pulses has a pulse width of 2ms to 50 ms.

Embodiment 74. The method of any of embodiments 1-3, comprising delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

Embodiment 75. The method of any of embodiments 71-74, wherein the user selects at least one of the first and second procedures, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first physical profile and the second metal implant has a second physical profile that is different from the first physical profile; the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

The method of embodiment 74, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; the first procedure includes a first duty cycle and the second procedure includes a second duty cycle different from the first duty cycle.

The method of embodiment 74, wherein: the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant; the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties; the first protocol includes a first amount of a therapeutic property and the second protocol includes a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

Embodiment 75.1. The method of embodiment 75, wherein the physical property comprises one of density (kg/m 3), electrical conductivity (S/m), relative permittivity, or thermal conductivity (W/(m.K)), specific heat (J/(kg.K)).

Embodiment 75.2 the method of any one of embodiments 71-75.1, wherein said therapeutic property comprises a total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Two doses Fixed time interval (minutes) between amounts (Δt) _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) One of them.

Embodiment 76. The method of any of embodiments 75-75.2 wherein the first procedure comprises a first period and the second procedure comprises a second period different from the first period.

Embodiment 77. The method of any of embodiments 75-76, wherein the first protocol comprises a first pulse width and the second protocol comprises a second pulse width different from the first pulse width.

Embodiment 78. The method of any one of embodiments 75-77, wherein: the first procedure includes applying a first duration of a plurality of pulses to a transmitter, and the second procedure includes applying a second duration of the plurality of pulses to the transmitter; the first duration is different from the second duration.

Embodiment 79 the method of any of embodiments 71-78, comprising delivering a plurality of AMF pulses to the metal implant to raise a temperature on a surface of the metal implant by less than 10 degrees celsius in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 80. The method of any of embodiments 71-79, comprising delivering a plurality of AMF pulses to the metal implant to induce a current of 50 to 3000A/cm 2 on a surface of the metal implant in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

Embodiment 81. The method of any of embodiments 71-80, comprising: sensing a parameter using at least one sensor; at least one of the duty cycle or period is changed in response to the sensed parameter.

Embodiment 81.1 the method of embodiments 75.2 and 81, comprising changing the treatment characteristic in response to the sensed parameter.

Embodiment 82. The method of embodiment 81 wherein the parameter comprises at least one of sound, temperature, resonance, energy, or a combination thereof.

Embodiment 83 the method of any one of embodiments 51-81, comprising administering a medication to a recipient of an AMF pulse within 1 week that the recipient receives the AMF pulse.

However, in some embodiments, no medication (e.g., antibiotics) is administered within 1 week after the patient receives a dose of AMF pulses.

Applicants observed anomalous results in which AMF exposure (i.e., 50 degrees celsius for 2 hours in a series of exposures) that produced low temperatures was toxic to the biofilm when combined with antibiotics, even though equivalent exposures from conduction heating in a temperature controlled water bath were ineffective. Applicants determined that embodiments that generate current from AMF contribute to sensitization of the biofilm to antibiotics. Applicant evaluates a short duration burst with a low duty cycle that will produce a high surface current but will leave enough time between bursts for heat to dissipate and not injure tissue. These provide unexpected therapeutic results. While the initial idea might be that biofilm reduction is largely a function of membrane temperature, applicants were able to determine that characteristics such as intermittently applied low pulse width can reduce biofilm when coupled with antibiotics without having to resort to high temperatures that can damage tissue surrounding the implant.

Thus, unexpected results appear. It is expected that low levels of energy together with antibiotics will not reduce biofilm.

Embodiment 84. The method of any of embodiments 51 to 83, comprising maintaining a temperature on the metal implant at 50-80 ℃ for more than 2 minutes.

Example 85. A method, comprising: administering an antibiotic to a patient; repeated administrations of short duration AMF exposure to a metal implant within a patient with sufficient cooling time between exposures to allow therapeutic thermal dose on the implant surface without concomitant increase in tissue thermal dose.

Embodiment 86 the method of embodiment 85 comprising adjusting at least one AMF parameter configured to allow a therapeutic thermal dose on the implant surface without concomitant increase in tissue thermal dose, wherein the at least one AMF parameter comprises at least one of a maximum temperature on the implant, a duration of application of AMF pulses to the patient, and an exposure per dose.

The ability to induce low temperature processing non-invasively while still producing significant current distinguishes embodiments from conventional systems/methods. Also, since lower temperature processing is desired, a lower power amplifier is required. Such an amplifier is more cost effective than larger amplifiers and should make smaller-scale clinics more affordable for the system.

Embodiments may include a user interface. Such a user interface may comprise a touch screen. Such an embodiment may operate as a stand-alone instrument to provide processing without any internet connection. However, the wireless connection may be used to download patient imaging data prior to processing. The embodiments may be used in a clinical setting (e.g., an outpatient or operating room). The system may be used initially by the orthopedic surgeon/orthopedic surgeon, but the operation may be delegated to the technician under their supervision and guidance. The technician may set up the system (e.g., power the device, download the appropriate patient records/images, and place the treatment transducer coil over or around the patient treatment area) and attend with the patient during treatment.

Embodiments may provide for a processing interrupt via logic circuitry if a high temperature signal is received from a safety sensor (e.g., an acoustic sensor that monitors the proximity of tissue to be processed to the implant) or if any anomalies are detected in the driving of the processing transducer coil. The anomalies include: coil short circuit (overcurrent), coil open circuit (undercurrent), gantry arm movement (gantry arm movement), and the like.

Embodiments of the user interface may include patient data registration fields, such as: name, patient ID number, date and time, menu of selecting implant, implant image with confirmation button. Thus, the procedure referred to herein may be selected based on the selection of certain types of implants having various physical parameters. The user interface may display the selected processing parameters.

The user interface may include an area for positioning information (e.g., operator input processes transducer coil position information). The screen may include: an image of the implant, a "correctly positioned" button (for the operator to confirm the correct position of the treatment transducer) and a "start treatment" button. The user interface may include an area for processing information. The screen may include: a display of selected processing parameters, a display of time of processing (progress bar), a "stop processing" button, and a "processing complete" indicator. The user interface may include an error information area (e.g., processing and other operations have ceased). The screen may indicate that: errors: "processing stop" and "error cause" (e.g., over temperature, operator stopped before a predetermined processing time, high/low processing power).

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Supplementary reference

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While the present invention has been described with respect to a limited number of embodiments, those skilled in the art will appreciate numerous modifications and variations therefrom. It is intended that the appended claims cover all such modifications and variations as fall within the true spirit and scope of this present invention.

Claims (20)

1. A system, comprising:

at least one Alternating Magnetic Field (AMF) transmitter configured to apply one or more AMF pulses to the metal implant; at least one function generator;

at least one processor; and

at least one machine readable medium having data stored thereon that, if used by the at least one processor, causes the at least one processor, the at least one function generator, and the at least one transmitter to perform operations comprising delivering a plurality of AMF pulses to the metal implant;

wherein each of the plurality of AMF pulses has a duty cycle of less than 1% and a period of 1ms to 60 seconds.

2. The system of claim 1, wherein the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT).

3. The system of claim 1, wherein each of the plurality of pulses has a pulse width of 1ms to 30 seconds.

4. The system of claim 3, wherein the operations comprise delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

5. The system of claim 1, wherein:

the at least one machine readable medium includes a first protocol configured for a first metal implant and a second protocol configured for a second metal implant;

the first metal implant has a first amount of physical properties and the second metal implant has a second amount of physical properties that is different from the first amount of physical properties;

the first protocol includes a first amount of a therapeutic property and the second protocol includes a second amount of a therapeutic property that is different from the first amount of the therapeutic property.

6. The system of claim 5, wherein the physical characteristics comprise at least one of density (kg/m 3), electrical conductivity (S/m), relative permittivity, or thermal conductivity (W/(m-K)), specific heat (J/(kg-K)).

7. The system of claim 6, wherein the therapeutic characteristic comprises a total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Fixed time interval (minutes) between two doses (Δt _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) At least one of (a) and (b).

8. The system of claim 7, wherein the therapeutic characteristic comprises a T of 50 to 80 °c _max Δt of 1 to 10 minutes _exp T of 5 to 120 minutes _dose T less than 10 seconds _exp 。

9. The system of claim 8, wherein the therapeutic characteristic comprises 3 to 50N _exp 、N _dose =1 to 7 and Δt _dose =10 to 30 hours.

10. The system of claim 6, wherein:

the first procedure includes a first period and the second procedure includes a second period different from the first period;

the first protocol includes a first pulse width and the second protocol includes a second pulse width different from the first pulse width.

11. The system of claim 10, wherein:

the first procedure includes applying a first duration of a first plurality of pulses to the transmitter, and the second procedure includes applying a second duration of a second plurality of pulses to the transmitter;

The first duration is different from the second duration.

12. The system of claim 1, wherein the operations comprise delivering the plurality of AMF pulses to the metal implant to raise a temperature on a surface of the metal implant by less than 10 degrees celsius in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

13. The system of claim 1, wherein the operations comprise delivering the plurality of AMF pulses to the metal implant to induce a current of 50 to 3000A/cm 2 on a surface of the metal implant in response to each of the plurality of pulses, each of the plurality of pulses having a duty cycle of less than 1% and a period of 1ms to 60 seconds.

14. The system of claim 1, comprising at least one sensor, wherein the operations comprise:

sensing a parameter using the at least one sensor;

at least one of the duty cycle or period is changed in response to the sensed parameter.

15. The system of claim 14, wherein:

the operations include changing a treatment characteristic in response to the sensed parameter;

The therapeutic properties include total number of doses (N _dose ) Length of exposure time (seconds) per pulse (t _exp ) The length of time between pulses of the dose (Δt _exp ) AMF pulse number per dose (N _exp ) Duration (hours) of each dose (duration of administration or t) _dose ) Fixed time interval (minutes) between two doses (Δt _dose ) To allow cooling of the metal implant, maximum target temperature (degrees celsius) of the metal implant (T _max ) At least one of (a) and (b).

16. A method, comprising:

using at least one Alternating Magnetic Field (AMF) transmitter, at least one function generator, and at least one processor to deliver a plurality of AMF pulses to the metal implant;

wherein each of the plurality of AMF pulses has a duty cycle of less than 1% and a period of 1ms to 60 seconds.

17. The method according to claim 16, wherein:

the plurality of AMF pulses have a magnetic field of no greater than 5 millitesla (mT);

each of the plurality of pulses has a pulse width of 2ms to 50 ms.

18. The method of claim 17, comprising delivering the plurality of AMF pulses to the metal implant for a duration of at least 30 minutes.

19. A method, comprising:

administering an antibiotic to a patient having an implanted metal implant;

Repeatedly applying Alternating Magnetic Field (AMF) exposures to the implant with a cooling time that occurs between the exposures, configured to both: (a) Allowing a therapeutic thermal dose on the implant surface, and (b) avoiding causing an excessive concomitant increase in tissue thermal dose;

wherein each of the exposures has a duty cycle of less than 1% and a period of 1ms to 60 seconds.

20. The method of claim 19, comprising adjusting at least one AMF parameter to adjust exposure to produce a cooling time configured to both: (a) Allowing a therapeutic thermal dose on the implant surface, and (b) avoiding causing an excessive concomitant increase in tissue thermal dose, wherein the at least one AMF parameter comprises at least one of a maximum temperature on the implant, a duration of an AMF pulse applied to the patient, and a number of exposures per exposure dose.