Synergistic use of anti-inflammatory ketorolac and gentamicin to target staphylococcal biofilms.

BACKGROUND: While antibiotics remain our primary tools against microbial infection, increasing antibiotic resistance (inherent and acquired) is a major detriment to their efficacy. A practical approach to maintaining or reversing the efficacy of antibiotics is the use of other commonly used therapeutics, which show synergistic antibacterial action with antibiotics. Here, we investigated the extent of antibacterial synergy between the antibiotic gentamicin and the anti-inflammatory ketorolac regarding the dynamics of biofilm growth, the rate of acquired resistance, and the possible mechanism of synergy. METHODS: Control (ATCC 12600, ATCC 35984) and clinical strains (L1101, L1116) of Staphylococcus aureus and Staphylococcus epidermidis with varying antibiotic susceptibility profiles were used in this study to simulate implant-material associated low-risk and high-risk biofilms in vitro. The synergistic action of gentamicin sulfate (GS) and ketorolac tromethamine (KT), against planktonic staphylococcal strains were determined using the fractional inhibitory concentration measurement assay. Nascent (6 h) and established (24 h) biofilms were grown on 316L stainless steel plates and the synergistic biofilm eradication activity was determined and characterized using adherent bacteria count, minimum biofilm eradication concentration (MBEC) measurement for GS, visualization by live/dead imaging, scanning electron microscopy, gene expression of biofilm-associated genes, and bacterial membrane fluidity assessment. RESULTS: Gentamicin-ketorolac (GS-KT) combination demonstrated synergistic antibacterial action against planktonic Staphylococci. Control and clinical strains showed distinct biofilm growth dynamics and an increase in biofilm maturity was shown to confer further resistance to gentamicin for both 'low-risk' and 'high-risk' biofilms. The addition of ketorolac enhanced the antibiofilm activity of gentamicin against acquired resistance in staphylococcal biofilms. Mechanistic studies revealed that the synergistic action of gentamicin-ketorolac interferes with biofilm morphology and subverts bacterial stress response altering bacterial physiology, membrane dynamics, and biofilm properties. CONCLUSION: The results of this study have a significant impact on the local administration of antibiotics and other therapeutic agents commonly used in the prevention and treatment of orthopaedic infections. Further, these results warrant the study of synergy for the concurrent or sequential administration of non-antibiotic drugs for antimicrobial effect.

Synergistic use of anti-inflammatory ketorolac and gentamicin to target staphylococcal biofilms.

Background: While antibiotics remain our primary tools against microbial infection, increasing antibiotic resistance (inherent and acquired) is a major detriment to their efficacy. A practical approach to maintaining or reversing the efficacy of antibiotics is the use of other commonly used therapeutics, which show synergistic antibacterial action with antibiotics. Here, we investigated the extent of antibacterial synergy between the antibiotic gentamicin and the anti-inflammatory ketorolac regarding the dynamics of biofilm growth, the rate of acquired resistance, and the possible mechanism of synergy. Methods: Control (ATCC 12600, ATCC 35984) and clinical strains (L1101, L1116) of S. aureus and S. epidermidis with varying antibiotic susceptibility profiles were used in this study to simulate implant-material associated low-risk and high-risk biofilms in vitro. The synergistic action of gentamicin sulfate (GS) and ketorolac tromethamine (KT), against planktonic staphylococcal strains were determined using the fractional inhibitory concentration measurement assay. Nascent (6hr) and established (24hr) biofilms were grown on 316 stainless steel plates and the synergistic biofilm eradication activity was determined and characterized using adherent bacteria count, MBEC measurement for GS, gene expression of biofilm-associated genes, visualization by live/dead imaging, scanning electron microscopy, and bacterial membrane fluidity assessment. Results: Gentamicin-ketorolac combination demonstrated synergistic antibacterial action against planktonic Staphylococci. Control and clinical strains showed distinct biofilm growth dynamics and an increase in biofilm maturity was shown to confer further resistance to gentamicin for both 'low-risk' and 'high-risk' biofilms. The addition of ketorolac enhanced the antibiofilm activity of gentamicin against acquired resistance in staphylococcal biofilms. Mechanistic studies revealed that the synergistic action of gentamicin-ketorolac interferes with biofilm morphology and subverts bacterial stress response altering bacterial physiology, membrane dynamics, and biofilm properties. Conclusion: The results of this study have a significant impact on the local administration of antibiotics and other therapeutic agents commonly used in the prevention and treatment of orthopaedic infections. Further, these results warrant the study of synergy for the concurrent or sequential administration of non-antibiotic drugs for antimicrobial effect.

Hydrogel device for analgesic drugs with in-situ loading and polymerization.

The high prevalence of opioid addiction and the shortcomings of systemic opioids has increased the pace of the search for alternative methods of pain management. The local delivery of pain medications has started to be used as a tool for pain management and to decrease the use of systemic opioids for these patients. Here, we explored an in-situ polymerizable hydrogel system for the local delivery of analgesics and nonsteroid anti-inflammatory drugs (NSAID) for orthopaedic applications. We synthesized a series of methacrylated oligomeric polyethylene glycol-co-lactic acid polymer using microwave radiation for the delivery of bupivacaine hydrochloride as an analgesic and ketorolac tromethamine as an NSAID. We determined drug elution and gel degradation profiles in vitro. Biocompatibility was assessed against osteoblasts in vitro and by histological analysis after subcutaneous implantation for 4 weeks in vivo. Intra-articular and systemic concentrations and pharmacokinetic parameters were estimated using a two-compartment pharmacodynamic model based on in-vitro elution profiles. This type of in-situ applicable hydrogels is promising for extending the local efficacy of pain medication and further reducing the need for opioids.

Dual-analgesic loaded UHMWPE exhibits synergistic antibacterial effects against Staphylococci.

Total joint arthroplasty is one of the most common surgeries in the United States, with almost a million procedures performed annually. Periprosthetic joint infections (PJI) remain the most devastating complications associated with total joint replacement. Effective antibacterial prophylaxis after primary arthroplasty could substantially reduce incidence rate of PJI. In the present study we propose to provide post-arthroplasty prophylaxis via dual-analgesic loaded ultra-high molecular weight polyethylene (UHMWPE). Our approach is based on previous studies that showed pronounced antibacterial activity of analgesic- and NSAID-loaded UHMWPE against Staphylococci. Here, we prepared bupivacaine/tolfenamic acid-loaded UHMWPE and assessed its antibacterial activity against Staphylococcus aureus and Staphylococcus epidermidis. Dual-drug loaded UHMWPE yielded an additional 1-2 log reduction of bacteria, when compared with single-drug loaded UHMWPE. Analysis of the drug elution kinetics suggested that the observed increase in antibacterial activity is due to the increased tolfenamic acid elution from dual-drug loaded UHMWPE. We showed that the increased fractal dimension of the drug domains in UHMWPE could be associated with increased drug elution, leading to higher antibacterial activity. Dual-analgesic loaded UHMWPE proposed here can be used as part of multi-modal antibacterial prophylaxis and promises substantial reduction in post-arthroplasty mortality and morbidity.

Nanoceria: Metabolic interactions and delivery through PLGA-encapsulation.

Cerium oxide nanoparticles (nanoceria) have recyclable antioxidative activity. It has numerous potential applications in biomedical engineering, such as mitigating damage from burns, radiation, and bacterial infection. This mitigating activity is analogous to that property of metabolic enzymes such as superoxide dismutase (SOD) and catalase - scavengers of reactive oxygen species (ROS). Therefore, nanoceria can protect cells from environmental oxidative stress. This therapeutic effect prompted studies of nanoceria and metabolic enzymes as a combination therapy. The activity and structure of SOD, catalase, and lysozyme were examined in the presence of nanoceria. A complementary relationship between SOD and nanoceria motivated the present work, in which we explored a method for simultaneous delivery of SOD and nanoceria. The biocompatibility and tunable degradation of poly(lactic-co-glycolic acid) (PLGA) made it a candidate material for encapsulating both nanoceria and SOD. Cellular uptake studies were conducted along with a cytotoxicity assay. The antioxidative properties of PLGA-nanoceria-SOD particles were verified by adding H2O2 to cell culture and imaging with fluorescent markers of oxidative stress. Our results suggest that PLGA is a suitable encapsulating carrier for simultaneous delivering nanoceria and SOD together, and that this combination effectively reduces oxidative stress in vitro.

Addressing prosthetic joint infections via gentamicin-eluting UHMWPE spacer.

AIMS: We propose a state-of-the-art temporary spacer, consisting of a cobalt-chrome (CoCr) femoral component and a gentamicin-eluting ultra-high molecular weight polyethylene (UHMWPE) tibial insert, which can provide therapeutic delivery of gentamicin, while retaining excellent mechanical properties. The proposed implant is designed to replace conventional spacers made from bone cement. METHODS: Gentamicin-loaded UHMWPE was prepared using phase-separated compression moulding, and its drug elution kinetics, antibacterial, mechanical, and wear properties were compared with those of conventional gentamicin-loaded bone cement. RESULTS: Gentamicin-loaded UHMWPE tibial components not only eradicated planktonic Staphylococcus aureus, but also prevented colonization of both femoral and tibial components. The proposed spacer possesses far superior mechanical and wear properties when compared with conventional bone cement spacers. CONCLUSION: The proposed gentamicin-eluting UHMWPE spacer can provide antibacterial efficacy comparable with currently used bone cement spacers, while overcoming their drawbacks. The novel spacer proposed here has the potential to drastically reduce complications associated with currently used bone cement spacers and substantially improve patients' quality of life during the treatment. Cite this article: Bone Joint J 2020;102-B(6 Supple A):151-157.

Synergistic antibacterial effects of analgesics and antibiotics against Staphylococcus aureus.

The local use of analgesics and antibiotics is common during the treatment of periprosthetic joint infection (PJI). The effect of nonantimicrobial drugs on antibacterial activity is underappreciated in clinical practice. This study focuses on the novel assessment of the combined antibacterial effects of commonly used analgesics and antibiotics against methicillin-sensitive Staphylococcus aureus (MSSA)-pathogen associated with most PJIs. We identified that bupivacaine/lidocaine and ketorolac/gentamicin combinations yielded fractional inhibitory concentration indices below 0.4, indicative of synergistic antibacterial effect. Time-kill curves were used for in-depth characterization of the synergy, and the obtained results demonstrated pronounced synergistic effects of bupivacaine/lidocaine and ketorolac/gentamicin combinations against MSSA.

Longitudinal Model of Periprosthetic Joint Infection in the Rat.

The majority of periprosthetic joint infections occur shortly after primary joint replacement (<3 months) and require the removal of all implant components for the treatment period (~4 months). A clinically relevant animal model of periprosthetic infection should, therefore, establish an infection with implant components in place. Here, we describe a joint replacement model in the rat with ultrahigh molecular weight polyethylene (UHMWPE) and titanium components inoculated at the time of surgery by methicillin-sensitive Staphylococcus aureus (S. aureus), which is one of the main causative microorganisms of periprosthetic joint infections. We monitored the animals for 4 weeks by measuring gait, weight-bearing symmetry, von Frey testing, and micro-CT as our primary endpoint analyses. We also assessed the infection ex vivo using colony counts on the implant surfaces and histology of the surrounding tissues. The results confirmed the presence of a local infection for 4 weeks with osteolysis, loosening of the implants, and clinical infection indicators such as redness, swelling, and increased temperature. The utility of specific gait analysis parameters, especially temporal symmetry, hindlimb duty factor imbalance, and phase dispersion was identified in this model for assessing the longitudinal progression of the infection, and these metrics correlated with weight-bearing asymmetry. We propose to use this model to study the efficacy of using different local delivery regimens of antimicrobials on addressing periprosthetic joint infections. Statement of clinical significance: We have established a preclinical joint surgery model, in which postoperative recovery can be monitored over a multi-week course by assessing gait, weight-bearing, and allodynia. This model can be used to study the efficacy of different combinations of implant materials and medication regimens. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1101-1112, 2020.

Delivery of bupivacaine from UHMWPE and its implications for managing pain after joint arthroplasty.

Total joint replacement is a widely used and successful surgical approach. Approximately 7 million US adults are currently living with a hip or knee replacement. However, the surgical procedures for total joint replacement are associated with significant postoperative pain, and current strategies do not adequately address this pain, which leads to patient dissatisfaction, reduced mobility, and increased risk of opioid addiction. We hypothesized that the ultra-high-molecular-weight polyethylene (UHMWPE) bearing surfaces used in total joint prosthetics could provide sustained release of the local anesthetic bupivacaine to provide relief from joint pain for an extended period of time after surgery. In this paper, we describe the production of bupivacaine-loaded UHMWPE (BPE) and measure the in vitro bupivacaine release kinetics of BPE. We found that bupivacaine could be released from BPE at clinically relevant rates for up to several days and that BPE possesses antibacterial effects. Therefore, bupivacaine-loaded UHMWPE is a promising material for joint replacement prostheses, and future studies will evaluate its safety and efficacy in in vivo models. STATEMENT OF SIGNIFICANCE: Total joint replacement is associated with significant pain and risk of infection. In our paper, we introduce bupivacaine-loaded ultra-high-molecular-weight polyethylene (BPE), which releases bupivacaine, a pain-treating drug, at doses comparable to currently used doses. Additionally, BPE inhibits the growth of infection-causing bacteria. Therefore, BPE may be able to reduce both postsurgical pain and risk of infection, potentially treating two of the most prominent complications associated with total joint replacement. To our knowledge, this is the first development of a material that can address both complications, and devices incorporating BPE would represent a significant advancement in joint arthroplasty prosthetics. More generally, the incorporation of therapeutic agents into ultra-high-molecular-weight polyethylene could impact many orthopedic procedures owing to its ubiquity.

Antimicrobial effect of anesthetic-eluting ultra-high molecular weight polyethylene for post-arthroplasty antibacterial prophylaxis.

Despite being a relatively safe surgery, total joint replacement is often associated with two major complications-severe post-operative pain and periprosthetic joint infection. Local sustained delivery of therapeutics to the surgical site has a potential to address these complications more effectively than current clinical approaches. Given that several analgesics were shown to possess antibacterial activity, we propose here to use analgesic-loaded ultra-high molecular weight polyethylene (UHMWPE) as a delivery vehicle to provide antimicrobial effect after an arthroplasty. Three commonly used anesthetics, lidocaine, bupivacaine, and ropivacaine, were analyzed in order to reveal the drug with the highest antibacterial activity against methicillin-sensitive Staphylococcus aureus. Having shown highest antibacterial activity in the bacterial susceptibility tests, bupivacaine was chosen to be incorporated into UHMWPE to provide antibacterial properties. Bupivacaine-loaded UHMWPE possessed moderate dose-dependent antimicrobial properties, decreasing the S. aureus proliferation rate by up to 70%. Biofilm formation was also substanitally inhibited during the first 9 h of culture as quantified by bacterial counts and SEM. This proof-of-concept study is first of its kind to demonstrate that analgesic-loaded UHMWPE can be used as part of a multimodal antimicrobial therapy. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

En Route to Practicality of the Polymer Grafting Technology: One-Step Interfacial Modification with Amphiphilic Molecular Brushes.

Surface modification with polymer grafting is a versatile tool for tuning the surface properties of a wide variety of materials. From a practical point of view, such a process should be readily scalable and transferable between different substrates and consist of as least number of steps as possible. To this end, a cross-linkable amphiphilic copolymer system that is able to bind covalently to surfaces and form permanently attached networks via a one-step procedure is reported here. This system consists of brushlike copolymers (molecular brushes) made of glycidyl methacrylate, poly(oligo(ethylene glycol) methyl ether methacrylate), and lauryl methacrylate, which provide the final product with tunable reactivity and balance between hydrophilicity and hydrophobicity. The detailed study of the copolymer synthesis and properties has been carried out to establish the most efficient pathway to design and tailor this amphiphilic molecular brush system for specific applications. As an example of the applications, we showed the ability to control the deposition of graphene oxide (GO) sheets on both hydrophilic and hydrophobic surfaces using GO modified with the molecular brushes. Also, the capability to tune the osteoblast cell adhesion with the copolymer-based coatings was demonstrated.

Albumin-Assisted Method Allows Assessment of Release of Hydrophobic Drugs From Nanocarriers.

Polymeric nanoparticles have been extensively studied as drug delivery vehicles both in vitro and in vivo for the last two decades. In vitro methods to assess drug release profiles usually utilize degradation of nanoparticles in aqueous medium, followed by the measurement of the concentration of the released drug. This method, however, is difficult to use for drugs that are poorly water soluble. In this study, a protocol for measuring drug release kinetic using albumin solution as the medium is described. Albumin is a major blood transport protein, which mediates transport of many lipid soluble compounds including fatty acids, hormones, and bilirubin. The use of a dialysis-based system utilizing albumin dialysate solution allows hydrophobic drug release from a diverse set of drug delivery modalities is demonstrated. The method using liposomes and PLGA nanoparticles as drug carriers, and two model hydrophobic drugs, 17ß-estradiol, and dexamethasone is validated.

In vitro study on the deterioration of polypropylene hernia repair meshes.

Despite the relative safety of the procedure, hernia repairs are often associated with chronic post-operative pain. Although this complication has been linked among others to mesh deterioration, details of the processes that lead this deterioration are still unknown. This work aims to bridge this gap by analyzing the chemical, physical and structural alterations in hernia repair meshes exposed to oxidative stress in vitro. Here, we developed a methodology to characterize effect of oxidation stress on structure and properties of polymeric hernia repair meshes. It was shown that structural changes in polypropylene meshes exposed to oxidative stress may involve formation of cross-links between the polymer chains, chain scissions, and hydrogen bonds between the carboxyl groups, which are formed in the material during the oxidation. These effects result in mesh stiffening, ultimately leading to chronic post-operative pain. Moreover, we demonstrated that Composix meshes are more vulnerable to the oxidative stress when compared with UltraPro meshes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2225-2234, 2018.

Anti-inflammatory coatings of hernia repair meshes: A pilot study.

The current prevalence of postoperative chronic pain from hernioplasty procedures employing polymer mesh is close to 30%. Most of the researchers agree that oxidative stress, resulting from the release of oxidants and enzymes during acute inflammatory response, is a key factor in the development of posthernioplasty complications. This results in both the decrease of the biomechanical properties and stiffening of the polymer fibers of the mesh, leading to chronic pain. Moreover, enhanced activity of inflammatory cells can lead to an excessive deposition of connective tissue around the implant. In this study polypropylene hernia repair meshes coated with vitamin E (α-tocopherol), a known antioxidant, were prepared and characterized. The absorption isotherm of vitamin E on the mesh was characterized and a release profile study yielded a promising results, showing sustained release of the drug over a 10-day period. An animal study was conducted, and histological analysis five weeks after implantation exhibited a reduced host tissue response for a modified mesh as compared to a plain mesh, as evidenced by a higher mature collagen to immature collagen ratio, as well as lower level of fatty infiltrates, neovascularization and fibrosis in the case of modified mesh. These results support the use of α-tocopherol as a potential coating in attempt to reduce the extent of postoperative inflammation, and thereby improve long-term outcomes of hernioplasty. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 589-597, 2018.

Antioxidant Activity of SOD and Catalase Conjugated with Nanocrystalline Ceria.

Interactions of nanoparticles with biological matter-both somatically and in nature-draw scientists' attention. Nanoparticulate systems are believed to be our saviors, acting as versatile drug delivery vehicles. However, they can also cause life-threatening bodily damage. One of the most important properties of nanocrystalline cerium dioxide is its antioxidant activity, which decreases the abundance of reactive oxygen species during inflammation. In this paper, we report on synergistic effects of inorganic cerium oxide (IV) nanoparticles conjugated with the antioxidative enzymes superoxide dismutase and catalase on scavenging oxygen and nitrogen radicals.

Novel Antibacterial Coating on Orthopedic Wires To Eliminate Pin Tract Infections.

Novel approaches to the prevention of microbial infections after the insertion of orthopedic external fixators are in great demand because of the extremely high incidence rates of such infections, which can reach up to 100% with longer implant residence times. Monolaurin is an antimicrobial agent with a known safety record that is broadly used in the food and cosmetic industries; however, its use in antimicrobial coatings of medical devices has not been studied in much detail. Here, we report the use of monolaurin as an antibacterial coating on external fixators for the first time. Monolaurin-coated Kirschner wires (K-wires) showed excellent antibacterial properties against three different bacterial strains, i.e., methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus epidermidis Approximately 6.0-log reductions of both planktonic and adherent bacteria were achieved using monolaurin-coated K-wires, but monolaurin-coated K-wires did not show any observable cytotoxicity with mouse osteoblast cell cultures. Overall, monolaurin-coated K-wires could be promising as potent antimicrobial materials for orthopedic surgery.