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. 2020 Feb 12;5(7):3165–3171. doi: 10.1021/acsomega.9b03072

Evaluation of Second-Generation Lipophosphonoxins as Antimicrobial Additives in Bone Cement

Eva Zborníková †,‡, Jiří Gallo §, Renata Večeřová ∥, Kateřina Bogdanová ∥, Milan Kolář ∥, Dragana Vítovská ⊥, Duy Dinh Do Pham †, Ondřej Pačes †, Viktor Mojr †, Hana Šanderová ⊥, Jitka Ulrichová #, Adéla Galandáková #, Drahomír Čadek ∇, Zdeněk Hrdlička ∇, Libor Krásný ⊥,*, Dominik Rejman †,*
PMCID: PMC7045315  PMID: 32118132

Abstract

graphic file with name ao9b03072_0006.jpg

Successful surgeries involving orthopedic implants depend on the avoidance of biofilm development on the implant surface during the early postoperative period. Here, we investigate the potential of novel antibacterial compounds—second-generation lipophosphonoxins (LPPOs II)—as additives to surgical bone cements. We demonstrate (i) excellent thermostability of LPPOs II, which is essential to withstand elevated temperatures during exothermic cement polymerization; (ii) unchanged tensile strength and elongation at the break properties of the composite cements containing LPPOs II compared to cements without additives; (iii) convenient elution kinetics on the order of days; and (iv) the strong antibiofilm activity of the LPPO II-loaded cements even against bacteria resistant to the medicinally utilized antibiotic, gentamicin. Thus, LPPOs II display promising potential as antimicrobial additives to surgical bone cements.

Introduction

Infections are feared complications of all indwelling orthopedic implants. These infections may result in the development of biofilms on the implant surface.1 The main causative agents are staphylococci followed by streptococci.2 A number of strategies have been developed to prevent these infections.3,4 A frequently used strategy is the usage of poly(methyl methacrylate) (PMMA) as a local carrier of antibiotics (ATB). ATB is released from the PMMA surface for several hours/days, targeting bacteria on its surface and in the near vicinity without potential systemic side effects.5 Gentamicin is the most commonly used antibiotic for these purposes. However, resistance to clinically used antibiotics, including gentamicin, is on the rise and poses a serious problem.6,7 Therefore, new compounds with improved antibacterial/antibiofilm characteristics and a low propensity for the development of resistant strains are highly desirable.

Several years ago, we reported the design and synthesis of new antibacterial compounds termed lipophosphonoxins (LPPOs). LPPOs are small amphiphilic molecules bearing positive charge(s). Their general structure (Figure 1) consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules (i)–(iii). First-generation LPPOs (LPPOs I) demonstrated excellent bactericidal activity against various Gram-positive species, including multiresistant strains such as vancomycin-resistant enterococci or methicillin-resistant Staphylococcus aureus. The minimum inhibitory concentration (MIC) values were in the 1–12 mg/L range, while their cytotoxic concentrations against human cell lines were above this range (IC50 60–100 mg/L).8,9

Figure 1.

Figure 1

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Lipophosphonoxins. The scheme in the left depicts the general structure of LPPOs. Examples of LPPOs I and II are shown in the middle and in the right. Compounds DR-6155 and DR-6180 were used in this study.

However, LPPO I compounds are ineffective against Gram-negative bacteria. By redesigning the iminosugar module, so that it bore more positive charges, we developed the second generation of LPPOs (LPPOs II) with increased efficacy (MIC < 1–6 mg/L) against Gram-positive species and an extended antibacterial activity range that now also includes serious Gram-negative pathogens, such as clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis.10 We have shown that at their bactericidal concentrations, LPPOs II act upon the cytoplasmic membrane of bacteria but not of eukaryotes. In vivo, LPPOs II do not inhibit synthesis of any of the cell macromolecules (DNA, RNA, protein, peptidoglycan, and membrane lipids). The only target of LPPOs II is the bacterial cytoplasmic membrane to which these compounds cause serious damage, resulting in efflux of the bacterial cytosol and cell disintegration. Employing model membranes (liposomes and black lipid membranes), we demonstrated that LPPOs II act by creating pores (with a conductance of up to 500 pS) in the membrane.10 Further, LPPOs II are well tolerated by live mice when administered orally (2000 mg/kg)10 and cause no skin irritation in rabbits (unpublished results). The general structure of LPPOs, an example of LPPOs I (compound DR-5026), and examples of LPPOs II (DR-6155 and DR-6180) (the bold numbers used throughout the text refer to LPPO structures) are depicted in Figure 1.

Finally and importantly, using several of the most potent LPPOs I and LPPOs II, we failed to select Bacillus subtilis, Enterococcus faecalis, or Streptococcus agalactiae strains resistant against DR-5026 and P. aeruginosa resistant to DR-6155, while resistant strains against known conventional antibiotics (rifampicin and ciprofloxacin, respectively) were readily developed or induced in control.8−10

In this proof-of-concept study, we evaluate selected LPPOs DR-6155 and DR-6180 as additives in PMMA bone cements, demonstrating their ability (i) to mix well with PMMA, (ii) to be unaffected by the increased temperature during PMMA polymerization, (iii) to minimally affect the tensile strength and elongation at the break properties of the composite material compared to only cement, (iv) to be continuously eluted from the bone cement for extended periods of time, and (v) to efficiently prevent biofilm formation upon elution.

Results and Discussion

Selection of LPPOs

We selected two LPPOs (DR-6155 and DR-6180) for subsequent studies as these compounds were the most effective ones against a range of Gram-positive and Gram-negative bacteria, including dangerous resistant pathogens (see Table 1 for MIC values) while displaying low cytotoxicity.10

Table 1. MIC Values against Selected Bacterial Strains for Compounds DR-6155 and DR-6180(10).

  MIC LPPO (mg/L)
bacterial strain DR-6180 DR-6155
CCM 4223 S. aureus 3.13 6.25
CCM 4224 E. faecalis 25 25
CCM 3954 E. coli 0.78 6.25
CCM 3955 P. aeruginosa 0.78 0.78
CCM 7221 Staphylococcus epidermidis 6.25 6.25
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Thermostability of LPPOs

PMMA bone cements increase their temperature up to 90 °C during the exothermic radical polymerization in dependence of the particular type of PMMA.11 Therefore, as an essential prerequisite, we first evaluated the ability of DR-6155 and DR-6180 to withstand increased temperatures during Bone cement R polymerization (78 °C according to the manufacturer). Therefore, we incubated two concentrations of each DR-6155 and DR-6180 dissolved in water (for details see the Experimental Section) at 80 °C for 4 and 8 h, respectively. The incubation times vastly surpass those required for Bone cement R polymerization (polymerization time ca. 10 min), but we wished to allow a generous margin to also evaluate the long-term thermostability of the tested compounds. Subsequently, we evaluated the integrity of the compounds by means of liquid chromatography–mass spectrometry (LC–MS). The LC–MS analysis revealed no degradation products even after the 8 h-long incubation, demonstrating excellent thermostability of the tested compounds (Figure S1).

Preparation of PMMA Bone Cement Loaded with LPPOs

In the next step, we designed and constructed apparatus for formation of PMMA bone cement pellets of uniform size (see the Experimental Section for details). LPPOs DR-6155 and DR-6180 mixed well with the cement and did not interfere with the polymerization process (see the next section). Importantly, the apparatus allowed creating pellets with identical weight and compaction.

Tensile Strength and Elongation at Break of Composite Cements

Tensile strength and elongation at break of composite cements containing DR-6180 were tested according to ISO 527 (for details see the Experimental Section). The results revealed no significant differences in both parameters (Figure 2) between composite cements containing up to 0.2 g of LPPO/10 g of cement and only cement, indicating that LPPOs did not negatively affect the polymerization process.

Figure 2.

Figure 2

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Tensile strength and elongation at break of composite cements. (A) Tensile strength and (B) Elongation at break of composite cements containing increasing amounts of DR-6180. P5-P9 are DR-6180: bone cement R ratio as defined in Table 1. The increasing amount of DR-6180 is indicated below the graphs. P10 (gray bars) is only bone cement R. The bars are averages from four independent experiments; the error bars represent ±SD.

Elution Kinetics of LPPOs from PMMA Bone Cement

Figure 3A shows two approaches that were used to determine the elution kinetics. The first approach consisted of withdrawing aliquots from the medium where the pellets were kept throughout the duration of the experiment; in the second approach, the pellets were transferred after indicated time points into fresh medium to mimic the exchange of the liquids surrounding the bone cement. For subsequent pilot experiments, we selected several ratios of the amount of LPPO (DR-6155 and DR-6180) per 10 g of PMMA (from 0.05 g of LPPO per 10 g of the PMMA bone cement to 0.2 g of LPPO per 10 g) to determine which ratio resulted in optimal, above MIC values of the eluted LPPO (Figure 3B). Interestingly, the elution was not linear with respect to the LPPO load in the bone cement. Then, 0.18 g of LPPO per 10 g of the PMMA bone cement was selected as the best combination. Importantly, this LPPO amount was still relatively small and should not compromise the mechanical properties of the bone cement, as shown in Figure 2.

Figure 3.

Figure 3

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Elution kinetics of LPPOs DR-6155 and DR-6180 from bone cement pellets. (A) A scheme of the two types of elution strategies. Upper panel: the pellet (yellow) was incubated in the same solution, and aliquots of the medium were withdrawn at time points for LC–MS; bottom panel: the pellet was transferred at time points into fresh solution. The solution from which the pellet had been transferred was used for LC–MS. The arrows indicate the flow of the experiments. (B) Elution of DR-6155 from bone cement after 480 min in physiological solution at 37 °C by the approach depicted in the upper panel of (A). The LPPO was mixed with the bone cement at indicated ratios. (C, D) The pellets with the tested LPPO (0.18 g of LPPO/10 g of cement) were immersed in physiological solution at 37 °C, and at time intervals, aliquots were withdrawn for LC–MS analysis, as depicted in the upper panel of (A). (E, F) The pellets with the tested LPPO (0.18 g of LPPO/10 g of cement) were immersed in physiological solution at 37 °C, and at time intervals, aliquots were withdrawn for LC–MS analysis. After each withdrawal, the pellets were transferred into fresh solution, as depicted in the lower panel of (A). (G) As in (E, F) but the elution time was prolonged and 0.18 g of LPPO/10 g of cement was used. The experiments were conducted in duplicate, the bars/dots show the averages, and the error bars show the range.

Then, we monitored the elution kinetics and its efficiency in more detail for DR-6155 and DR-6180. We analyzed the amount of eluted DR-6155 and DR-6180 by LC–MS. Figure 3C,D shows the elution kinetics of DR-6155 and DR-6180 over 8 h without pellet transfer into a fresh solution. The graph shows that DR-6155 and DR-6180 were eluted till the end of the experiment, with a rapid initial spike and a more gradual tail. Next, we performed experiments with pellet transfer. In the first round of these experiments that were limited to 24 h, DR-6155 and DR-6180 were continuously eluted over the monitored time period (Figure 3E,F). As with the first type of experiment, we noticed that the kinetics appeared to be biphasic: a strong initial spike followed by more gradual elution. Next, we extended the time period till 8 days (with pellet transfer; performed only for DR-6180; Figure 3G) and observed a similar type of kinetics but the compound was continuously eluted even over the prolonged time period. We also noticed that DR-6155 was consistently being eluted more efficiently than DR-6180. It could be explained by the higher polarity and aqueous solubility of compound DR-6155.

Antibiofilm Activity of LPPO-Loaded Cements

Finally, we tested the ability of LPPOs DR-6155 and DR-6180 to prevent biofilm formation on PMMA bone cements. We used LPPOs DR-6155 and DR-6180 in a range of concentrations (from 0.05 to 0.2 g of LPPO DR-6155 or DR-6180 in 10 g of PMMA Bone cement R). As a control, we used pellets with gentamicin (Palacos R+G). This particular bone cement contains 0.9 g of gentamicin sulfate per 40 g of PMMA bone cement (LPPOs were in the form of hydrochlorides). We used standard reference bacterial strains (E. faecalis CCM 4224, S. aureus CCM 4223, E. coli CCM 3954, and P. aeruginosa 3955) from the Czech Collection of Microorganisms (CCM), Faculty of Science, Masaryk University, Brno. Furthermore, the reference bacterial strain S. epidermidis CCM 7221 bearing the ica operon (polysaccharide intercellular adhesion12), which is frequently used as a standard for biofilm production, was included in the study.

The PMMA loaded with DR-6155 at 0.2 g/10 g of cement showed complete inhibitory effects against biofilm formation for all tested bacterial strains, i.e., S. epidermidis, S. aureus, E. faecalis, E. coli, and P. aeruginosa (Figure 4). In the case of DR-6180 also the biofilm formation was completely inhibited with tested bacterial strains at the same concentration, with the exception of P. aeruginosa. Lower LPPO concentrations displayed altered antibacterial efficiencies that were specific for each LPPO. Controls without antibiotics showed, as expected, no biofilm formation inhibition, whereas the gentamicin-containing commercial bone cement functioned on all tested bacterial strains with the exception of gentamicin-resistant S. epidermidis.

Figure 4.

Figure 4

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Biofilm formation in the presence of pellets from bone cement loaded with the selected LPPO II. The biofilm formation was evaluated as bacterial counts of bacteria (CFU/mL) released from the pellets by gentle sonication and subsequently grown on Mueller–Hinton Agar plates. The graph shows the experiment performed in duplicate. The pellets were either without any additive (P10) or with LPPO DR-6155 or DR-6180 or gentamicin. The exact ratios of added substances to the bone cement were as follows: P1—DR-6155 concentration 0.05 g/10 g PMMA, P2—DR-6155 concentration 0.15 g/10 g PMMA, P3—DR-6155 concentration 0.18 g/10 g PMMA, P4—DR-6155 concentration 0.2 g/10 g PMMA, P5—DR-6180 concentration 0.05 g/10 g PMMA, P6—DR-6180 concentration 0.15 g/10 g PMMA, P7—DR-6180 concentration 0.18 g/10 g PMMA, P8—DR-6180 concentration 0.2 g/10 g PMMA, P9 (Gent.)—gentamicin sulfate concentration 0.225 g/10 g PMMA, and P10—PMMA.

Conclusions

Biofilm formation is a serious problem in prosthetic joint infections,13 and various strategies for “anti-infective” implants were tested in the past.1,3,4,14 An urgent need exists for compounds to be used as additives to PMMA with low probability for resistance development.15,16 In this in vitro study, we characterized two new compounds, DR-6155 and DR-6180, for their potential as novel bone cement additives and compared these two compounds with gentamicin. Gentamicin primarily binds to the 30S ribosomal subunit and causes misincorporation of amino acids, causing protein aggregation and cell death.17 However, resistance to gentamicin arises easily, mostly through its modification (acetylation, phosphorylation, and adenylylation of hydroxy groups) in the bacterial cell and by efflux mechanisms.18

First, we showed that LPPOs DR-6155 and DR-6180 are highly thermostable, which is necessary for maintenance of their antibacterial activity after the exothermic phase of the PMMA polymerization process.19 Second, various amounts of DR-6180 were mixed with the bone cement and tested for tensile strength and elongation at break properties. The results of both tests revealed virtually no adverse effect of the presence of DR-6180 in the cement. Further, various concentrations of DR-6155 and DR-6180 were subjected to in vitro testing of their elution period and antibacterial efficacy. We found that their elution kinetics resembled the elution of known antibiotics.20 An initial spike was observed within a few hours after the application, followed by a more gradual prolonged elution on the order of days. This aligns well with the potential therapeutic use, where the elevated initial concentration kills existing bacteria, and this is followed by the elution of decreased, but still sufficient, amounts of the compounds providing the antibacterial milieu for an extended period of time.21 We speculate that the initial burst is the result of the contact between the fluid and the surface of PMMA pellets. Similar to ATB-laden PMMA, this could be followed by a release of DR-6155 and DR-6180 from deeper layers of the cement caused by elution via tiny pores on the cement surface. We could expect increased porosity of our PMMA pellets compared to vacuum-mixed PMMA.

Previously, DR-6155 and DR-6180 were tested for their antimicrobial properties against planktonic cells.10 Here, we demonstrated their efficacy against biofilm formation by the most frequent pathogens associated with prosthetic joint infections, coagulase-negative Staphylococci and S. aureus. Importantly, DR-6155 and DR-6180, unlike gentamicin (Figure 4), were also effective against gentamicin-resistant S. epidermidis, an aggressive biofilm former and causative agent of various infections, including those related to implanted medical devices.22,23

Finally and importantly, the aggressive mode of action of LPPOs DR-6155 and DR-6180 against the bacterial membrane (fast membrane permeabilization and cell lysis) and the apparent lack of resistant strains10 compares well with other antibiotics, such as the clinically used gentamicin or even the newly discovered optimized arylomycins.24 Based on these observations and results presented in this communication, we conclude that the tested second-generation LPPOs display excellent potential as antibacterial additives to bone cement to prevent bacterial prosthetic joint infections and biofilm formation. Studies on animal models evaluating LPPOs are currently in progress and will be reported in due time.

Experimental Section

Synthesis of Lipophosphonoxins

LPPOs DR-6155 and DR-6180 were synthesized according to a literature procedure.10

Thermostability of LPPOs

For determination of the stability of compounds DR-6155 and DR-6180 at evaluated temperatures, the following setup was used: analytes were dissolved in water at concentrations 1 mg/mL and 20 μg/mL and were shaken at 80 °C for 8 h; during this time, the compound stability was checked using the same high performance liquid chromatography (HPLC) method as that used for the analysis of elution kinetics (vide supra, LC–MS Analysis of Elution Kinetics).

Preparation of LPPO-Loaded Bone Cement Pellets

First, a device for producing uniformly sized bone cement pellets was designed and built by the IOCB Development Center. Since there is no standard for pellet size, we selected the one shown in Figure 5.

Figure 5.

Figure 5

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Device for producing uniformly sized bone cement pellets ((A) poly(tetrafluoroethylene) (PTFE) plate; (B) round holes in the shape of the resulting pellets; (C) pins; (D) metal spacers; (E) screws and nuts).

The mold consists of a poly(tetrafluoroethylene) (PTFE) plate (A) with round holes (B) in the shape of the pellets. The PTFE material has an advantage of low adhesion, leading to easy separation of the product (pellets). There are pins (C) that go through the plate to the bottoms of all holes. The pins are strongly held in the second plate. Next, there are spacers (D) between both plates. The whole assembly is held together by screws and nuts (E). When the spacers (D) are removed, a space is formed between the plates. If the screws become tightened, both plates go closer and the pins (C) go through the plate pushing out the pellets. PTFE main plate (A) and bottom plate were milled using a CNC machine. Ballmill cutter was used to achieve a proper shape and smooth surface of sample holes. Spacers (D) were cut by an Nd/YAG laser from a stainless steel sheet. Pins (C) were standard commercial stainless steel nonhardened pins. Special screws (E) were milled using a CNC machine from aluminum alloy 6061.

Second, the PMMA powder (Bone cement R; Zimmer Biomet) was mixed with LPPOs (in the form of Tris-hydrochloride) with pharmaceutical accuracy at the Hospital Pharmacy. The ratios of LPPOs DR-6155 and DR-6180 to the PMMA powder are shown in Table 2. Then, the mixture of PMMA polymer and LPPO was mixed with the liquid monomer manually in the aseptic operating room of the Department of Orthopedics under standard surgical conditions. LPPO-loaded PMMA was then applied into the above-described device to produce pellets. Pellets made of Palacos R+G (0.9 g of gentamicin sulfate (0.5 g gentamicin base) per 40 g PMMA; Heraeus Medical GmbH) were produced similarly.

Table 2. Ratios of LPPOs DR-6155 and DR-6180 to the PMMA Powder.

no. LPPO LPPO/PMMA ratio (g/g)
P-1 DR-6155 0.05/10
P-2 DR-6155 0.15/10
P-3 DR-6155 0.18/10
P-4 DR-6155 0.20/10
P-5 DR-6180 0.05/10
P-6 DR-6180 0.15/10
P-7 DR-6180 0.18/10
P-8 DR-6180 0.20/10
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Elution Kinetics

Experiment A

The pellet was incubated in 10 mL of physiological solution in a 50 mL Falcon tube in a shaker at 37 °C. At appropriate time points (0, 60, 120, 240, and 480 min), 50 μL of solution was taken and directly analyzed by LC–MS.

Experiment B

The pellet was incubated in 1 mL of physiological solution in a 2 mL Eppendorf tube in a shaker at 37 °C. At appropriate time points (1, 2, 4, 8, and 24 h or 1, 2, 4, and 8 days), the pellet was transferred to a 2 mL Eppendorf tube with 1 mL of fresh physiological solution and the original solution was analyzed.

LC–MS Analysis of Elution Kinetics

All experiments were analyzed on a ultra-performance liquid chromatography (UPLC; Acquity, H-class; Waters) instrument with both mass spectrometric (Xevo qTOF; Waters) and photodiode array (Waters) detectors using a C18 column (Acquity UPLC BEH, 2.1 × 50 mm2, 1.7 μm; Waters). Injection volume was 2 μL and gradient conditions were as follows: 0–0.5 min 20% B, increased to 95% B in the 4th min, held till the 5th min, equilibrated for 2 min, where mobile phase A was 0.05% formic acid and B was 0.05% formic acid in acetonitrile. Mass detection was realized by positive electrospray ionization in scan mode from 300 to 900 m/z; during the first 0.3 min, the eluent was disposed to the waste.

Quantification was done by external standard calibration based on a UV signal at 260 nm. Eluted products were identified by retention times and confirmed with mass spectra.

In the case of repetitive solution withdrawal (Experiment A), the total volume of the physiological solution was 10 mL. At each time point, 50 μL of solution was taken and directly analyzed. In the case where pellets were transferred at each time point in the fresh solution (Experiment B), the volume of the physiological solution was 1 mL. This solution was evaporated to dryness and dissolved in 50 μL of HPLC grade water.

Testing Antibiofilm Activity of LPPO-Loaded Cements

To test the inhibition of biofilm formation on the surface of bone cement with added LPPOs (DR-6155 and DR-6180) in a range of concentrations from 0.05 to 0.2 g of tested LPPO/10 g of cement, standard reference bacterial strains (E. faecalis CCM 4224, S. aureus CCM 4223, E. coli CCM 3954, and P. aeruginosa CCM 3955) from the Czech Collection of Microorganisms (CCM), Faculty of Science, Masaryk University, Brno, were used. Furthermore, the reference bacterial strain, S. epidermidis CCM 7221, with positive biofilm formation and the ica operon was included in the study.

Bone cement R without an antibiotic or LPPO and bone cement with gentamicin (Palacos R+G) served as negative and positive controls. The proper numbers of pellets (two pieces of each type per each tested bacterium) were placed into culture plate wells (Thermo Fisher Scientific). Subsequently, 2 mL of a culture medium (BHI—Brain Heart Infusion; Hi-Media) with the tested bacterial strains was added into the culture plate wells. The culture plates were placed on a shaker (BIOSAN) in a thermostat and cultured at 35 °C for 24 h.

After culturing, cement beads were washed in 1 mL of distilled water and placed into microcentrifuge tubes with 1 mL of culture medium (BHI). Then, the tubes were vortexed, sonicated in a water bath for 2 min (Sonorex RK156, Fischer Scientific) to release the formed biofilm, and vortexed again. The tube contents with sonicated cement beads were diluted and inoculated onto Mueller–Hinton agar (TRIOS) and cultured at 35 °C for 24 h. Subsequently, bacterial count was semiquantitatively estimated. The limit of detection was 100 cfu/mL.

Fabrication of Dumbbell Specimens

First, a device for producing uniformly sized bone cement Dumbbell specimens (Figure 6) was designed and built by the IOCB Development Center. Both PTFE main plates (A; the top and the bottom) were milled using a CNC machine. Spacer (B) was cut with an Nd/YAG laser from a stainless steel sheet.

Figure 6.

Figure 6

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Device for producing uniformly sized bone cement Dumbbell specimens: top PTFE main plate (A1), bottom PTFE main plate (A2), and stainless steel spacer (B).

Second, the PMMA powder (Bone cement R; Zimmer Biomet) was mixed with LPPOs (in the form of Tris-hydrochloride) with pharmaceutical accuracy at the Hospital Pharmacy. The ratios of LPPOs DR-6155 and DR-6180 to the PMMA powder are shown in Table 2. Then, the mixture of PMMA polymer and LPPO was mixed with the liquid monomer manually in the aseptic operating room of the Department of Orthopedics under standard surgical conditions. LPPO-loaded PMMA was then applied into the above-described device to produce Dumbbell specimens. The dimensions of the specimens were as follows: width 2 mm, nominal thickness 2 mm, and length of the specimen between grips 10 mm.

Tensile Tests

Tensile tests of the prepared materials were performed on an Instron 3365 according to ISO 527.25 Tensile strength and elongation were evaluated. Pneumatic grips with a speed of tearing of 1 mm/min were used. Elongation was evaluated from the crosspiece movement.

Acknowledgments

This work was supported by the funds from the Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, v.v.i. to D.R., and Czech Ministry of Health (Grant No. 17-29680A to D.R., J.G., and L.K.).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03072.

  • Thermal stability of LPPOs; monoisotopic chromatogram (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b03072_si_001.pdf (256.5KB, pdf)

References

  1. Thet N. T.; Wallace L.; Wibaux A.; Boote N.; Jenkins A. T. A. Development of a mixed-species biofilm model and its virulence implications in device related infections. J. Biomed. Mater. Res., Part B 2019, 107, 129–137. 10.1002/jbm.b.34103. [DOI] [PubMed] [Google Scholar]
  2. Peel T. N.; Cheng A. C.; Buising K. L.; Choong P. F. Microbiological aetiology, epidemiology, and clinical profile of prosthetic joint infections: are current antibiotic prophylaxis guidelines effective?. Antimicrob. Agents Chemother. 2012, 56, 2386–2391. 10.1128/AAC.06246-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gallo J.; Holinka M.; Moucha C. S. Antibacterial surface treatment for orthopaedic implants. Int. J. Mol. Sci. 2014, 15, 13849–13880. 10.3390/ijms150813849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gallo J.; Panacek A.; Prucek R.; Kriegova E.; Hradilova S.; Hobza M.; Holinka M. Silver Nanocoating Technology in the Prevention of Prosthetic Joint Infection. Materials 2016, 9, 337 10.3390/ma9050337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Miola M.; Bistolfi A.; Valsania M. C.; Bianco C.; Fucale G.; Verne E. Antibiotic-loaded acrylic bone cements: an in vitro study on the release mechanism and its efficacy. Mater. Sci. Eng., C 2013, 33, 3025–3032. 10.1016/j.msec.2013.03.032. [DOI] [PubMed] [Google Scholar]
  6. Baker S.; Thomson N.; Weill F. X.; Holt K. E. Genomic insights into the emergence and spread of antimicrobial-resistant bacterial pathogens. Science 2018, 360, 733–738. 10.1126/science.aar3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Marston H. D.; Dixon D. M.; Knisely J. M.; Palmore T. N.; Fauci A. S. Antimicrobial Resistance. J. Am. Med. Assoc. 2016, 316, 1193–1204. 10.1001/jama.2016.11764. [DOI] [PubMed] [Google Scholar]
  8. Rejman D.; Rabatinova A.; Pombinho A. R.; Kovackova S.; Pohl R.; Zbornikova E.; Kolar M.; Bogdanova K.; Nyc O.; Sanderova H.; Latal T.; Bartunek P.; Krasny L. Lipophosphonoxins: New Modular Molecular Structures with Significant Antibacterial Properties. J. Med. Chem. 2011, 54, 7884–7898. 10.1021/jm2009343. [DOI] [PubMed] [Google Scholar]
  9. Panova N.; Zbornikova E.; Simak O.; Pohl R.; Kolar M.; Bogdanova K.; Vecerova R.; Seydlova G.; Fiser R.; Hadravova R.; Sanderova H.; Viovska D.; Sikova M.; Latal T.; Lovecka P.; Barvik I.; Krasny L.; Rejman D. Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins. PLoS One 2015, 10, e0145918 10.1371/journal.pone.0145918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Seydlová G.; Pohl R.; Zbornikova E.; Ehn M.; Simak O.; Panova N.; Kolar M.; Bogdanova K.; Vecerova R.; Fiser R.; Sanderova H.; Vitovska D.; Sudzinova P.; Pospisil J.; Benada O.; Krizek T.; Sedlak D.; Bartunek P.; Krasny L.; Rejman D. Lipophosphonoxins II: Design, Synthesis, and Properties of Novel Broad Spectrum Antibacterial Agents. J. Med. Chem. 2017, 60, 6098–6118. 10.1021/acs.jmedchem.7b00355. [DOI] [PubMed] [Google Scholar]
  11. Anagnostakos K.; Fink B. Antibiotic-loaded cement spacers - lessons learned from the past 20 years. Expert Rev. Med. Devices 2018, 15, 231–245. 10.1080/17434440.2018.1435270. [DOI] [PubMed] [Google Scholar]
  12. Cafiso V.; Bertuccio T.; Santagati M.; Campanile F.; Amicosante G.; Perilli M. G.; Selan L.; Artini M.; Nicoletti G.; Stefani S. Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin. Microbiol. Infect. 2004, 10, 1081–1088. 10.1111/j.1469-0691.2004.01024.x. [DOI] [PubMed] [Google Scholar]
  13. Gundtoft P. H. Prosthetic Joint Infection following Total Hip Arthroplasty - Incidence, Mortality and Validation of the Diagnosis in the Danish Hip Arthroplasty Register. Dan. Med. J. 2017, 64, B5397. [PubMed] [Google Scholar]
  14. Fillingham Y.; Jacobs J. Bone grafts and their substitutes. Bone Joint J. 2016, 98-B, 6–9. 10.1302/0301-620X.98B.36350. [DOI] [PubMed] [Google Scholar]
  15. Tan H. L.; Lin W. T.; Tang T. T. The use of antimicrobial-impregnated PMMA to manage periprosthetic infections: controversial issues and the latest developments. Int. J. Artif. Organs 2012, 35, 832–839. 10.5301/ijao.5000163. [DOI] [PubMed] [Google Scholar]
  16. Gálvez-López R.; Pena-Monje A.; Antelo-Lorenzo R.; Guardia-Olmedo J.; Moliz J.; Hernandez-Quero J.; Parra-Ruiz J. Elution kinetics, antimicrobial activity, and mechanical properties of 11 different antibiotic loaded acrylic bone cement. Diagn. Microbiol. Infect. Dis. 2014, 78, 70–74. 10.1016/j.diagmicrobio.2013.09.014. [DOI] [PubMed] [Google Scholar]
  17. Krause K. M.; Serio A. W.; Kane T. R.; Connolly L. E. Aminoglycosides: An Overview. Cold Spring Harbor Perspect. Med. 2016, 6, a027029 10.1101/cshperspect.a027029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dowding J. E. Mechanisms of gentamicin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 1977, 11, 47–50. 10.1128/AAC.11.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vaishya R.; Chauhan M.; Vaish A. Bone cement. J. Clin. Orthop. Trauma 2013, 4, 157–163. 10.1016/j.jcot.2013.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Webb J. C.; Gbejuade H.; Lovering A.; Spencer R. Characterisation of in vivo release of gentamicin from polymethyl methacrylate cement using a novel method. Int. Orthop. 2013, 37, 2031–2036. 10.1007/s00264-013-1914-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Anagnostakos K.; Meyer C. Antibiotic Elution from Hip and Knee Acrylic Bone Cement Spacers: A Systematic Review. BioMed Res. Int. 2017, 2017, 1–7. 10.1155/2017/4657874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Otto M. Molecular basis of Staphylococcus epidermidis infections. Semin. Immunopathol. 2012, 34, 201–214. 10.1007/s00281-011-0296-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Le K. Y.; Dastgheyb S.; Ho T. V.; Otto M. Molecular determinants of staphylococcal biofilm dispersal and structuring. Front. Cell. Infect. Microbiol. 2014, 4, 167 10.3389/fcimb.2014.00167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith P. A.; Koehler M. F. T.; Girgis H. S.; Yan D.; Chen Y.; Chen Y.; Crawford J. J.; Durk M. R.; Higuchi R. I.; Kang J.; Murray J.; Paraselli P.; Park S.; Phung W.; Quinn J. G.; Roberts T. C.; Rouge L.; Schwarz J. B.; Skippington E.; Wai J.; Xu M.; Yu Z.; Zhang H.; Tan M. W.; Heise C. E. Optimized arylomycins are a new class of Gram-negative antibiotics. Nature 2018, 561, 189–194. 10.1038/s41586-018-0483-6. [DOI] [PubMed] [Google Scholar]
  25. ISO 527. Plastics—Determination of Tensile Properties; International Organization for Standardization: Genéve, 2012; p 23.

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