Biomaterial-associated Infections Research Articles

Inhibition of bacterial adhesion and biofilm formation by dual functional textured and nitric oxide releasing surfaces.

Microbial infection remains a significant barrier to development and implementation of advanced blood-contacting medical devices. Clearly, determining how to design and control material properties that can reduce microbial infection is a central question to biomaterial researchers. In separate prior studies, physical topographic surface modification or nitric oxide (NO) release has been demonstrated to each be an effective approach to inhibit and control bacterial adhesion and biofilm formation on polymeric surfaces. Such approaches can prevent biomaterial-associated infection without causing antibiotic resistance of the bacterial strain. However, efficiency of antimicrobial properties of each approach is still limited and far from sufficient for widespread clinical use. This work successfully integrates both techniques and applies them to a polyurethane (PU) biomaterial surface that bears dual functions, surface topographic modification and NO release. The former reduces the surface contact area and changes surface wettability, resulting in reduction of bacterial adhesion, and NO release further inhibits bacteria growth. Such dual functionalized surfaces provide a synergistic effect on inhibition of Staphylococcus epidermidis bacterial adhesion that is significantly greater than the inhibition of bacterial adhesion achieved by either single treatment approach alone. Furthermore, longer-term experiments demonstrate that the dual functionalized surfaces can inhibit biofilm formation for >28days. The success of this work provides a practical approach to improve the biocompatibility of current biomaterials and thereby reduce the risk of pathogenic infection.

Dual-functional bone implants with antibacterial ability and osteogenic activity.

A growing number of biomaterial-associated infections cause bone implant failures in early and long-term applications. In this regard, a calcium silicate-gelatine composite bone implant with high strength and superior osteogenic activity was coated with a layer of Ag, chitosan polysaccharide (CS) or water-soluble chitosan oligosaccharide (COS) as a bactericidal agent. The influences of surface modifications to the bone implants on phase composition, microstructure, antibacterial effectiveness, and osteogenic activity in vitro were evaluated. Experimental results revealed the presence of the coating on the implant surface using a simple deposition technique. The in vitro antibacterial evaluation indicated that the antimicrobial effectiveness of the Ag coating against Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) was inferior to the 0.4% CS coating, but comparable to those of 0.2% CS and 0.4% COS coatings after 48 h of culture. CS presented a greater bactericidal effect than COS, which was bacteria-independent. CS and COS coatings had no significant cytotoxicity towards L929 cells at coating concentrations of 0.1%, 0.2%, and 0.4%, except for the cells exposed to the 0.4% CS coating, while the 0.004% Ag coating remarkably produced cytotoxicity. The assays of cell functions consistently showed significantly higher osteogenic activity of MG63 cells grown on CS and COS-coated surfaces by increased attachment, proliferation, alkaline phosphatase, osteocalcin, and calcium deposits production, except for the 0.4% CS coating, in comparison with those on the Ag coated surface. It was concluded that, taking antibacterial ability and osteogenic activity into account, 0.2% CS-coated and 0.4% COS-coated calcium silicate-gelatine composite bone implants had a large potential to be used in bone grafts and fracture fixation devices.

The Level of Inflammatory Cytokines and Antimicrobial Peptides after Composite Material Implantation and Contamination with Bacterial Culture

As biomaterial implantation is a traumatic process, especially if the implanted object becomes contaminated by bacteria, it can cause a significant increase in the production of inflammatory cytokines. An increase in the level of inflammatory cytokines can be used as a marker of biomaterial associated infection (BAI) in surrounding tissues. To prevent BAI and production of inflammatory cytokines, biomaterials with antibiotics should be used, in particular biomaterials with prolonged release of antibiotics. In this in vivo study, the level of inflammatory cytokines (interleukine – 10 (IL-10), beta-defensin-2 and tumor necrosis factors (TNF-alpha)) was determined in surrounding tissues after composite material implantation in vivo and wound contamination with Staphylococcus epidermidis (S. epidermidis) or Pseudomonas aeruginosa (P. aeruginosa). The results show that the level of inflammatory cytokines is normal in surrounding tissues after implantation of biomaterials with prolonged release of antibiotics. Biomaterials with rapid release of antibiotics also show normal levels of inflammatory cytokines. The level of inflammatory cytokines increases in cases if biomaterials without antibiotics are implanted in vivo, thus being an indication of inflammation process and BAI.

Co-immobilization of Palm and DNase I for the development of an effective anti-infective coating for catheter surfaces.

Biomaterial-associated infections, in particular, catheter-associated infections (CAI) are a major problem in clinical practice due to their ability to resist antimicrobial treatment and the host immune system. This study aimed to co-immobilize the antimicrobial lipopeptide Palm and the enzyme DNase I to introduce both antimicrobial and anti-adhesive functionalities to polydimethylsiloxane (PDMS) material, using dopamine chemistry. Surface characterization confirmed the immobilization of both compounds and no leaching of Palm from the surfaces for up to 5days. Co-immobilization of both agents resulted in a bifunctional coating with excellent surface antimicrobial and anti-biofilm properties against both Staphylococcus aureus and Pseudomonas aeruginosa. The modified surfaces demonstrated superior biocompatibility. To better discriminate co-adhesion of both species on modified surfaces, PNA FISH (Fluorescence in situ hybridization using peptide nucleic acid probes) was employed, and results showed that P. aeruginosa was the dominant organism, with S. aureus adhering afterwards on P. aeruginosa agglomerates. Furthermore, Palm immobilization exhibited no propensity to develop bacterial resistance, as opposite to the immobilization of an antibiotic. The overall results highlighted that co-immobilization of Palm and DNase I holds great potential to be applied in the development of catheters. Catheter-associated infections (CAI) are the most common hospital-acquired infections worldwide. Several coating strategies have been proposed to fight these infections but most of them present some important limitations, including the emergence of resistant bacteria and toxicity concerns. The present work describes a two-step polydopamine-based surface modification strategy to successfully co-immobilize an antimicrobial peptide (Palm) and an enzyme targeting an important component of biofilm matrix (DNase I). This immobilization approach imparted polydimethylsiloxane surfaces with both anti-adhesive and antimicrobial properties against the adhesion of relevant bacteria as single and dual-species, with excellent stability and biocompatible and anti-biofilm properties, holding, therefore, great potential in the development of catheters able to prevent CAI.

Antibacterial Efficiency of Hydroxyapatite Biomaterials with Biodegradable Polylactic Acid and Polycaprolactone Polymers Saturated with Antibiotics / Bionoārdāmu Polimēru Saturošu Un Ar Antibiotiskajām Vielām Piesūcinātu Biomateriālu Antibakteriālās Efektivitātes Noteikšana

Abstract Infections continue to spread in all fields of medicine, and especially in the field of implant biomaterial surgery, and not only during the surgery, but also after surgery. Reducing the adhesion of bacteria could decrease the possibility of biomaterial-associated infections. Bacterial adhesion could be reduced by local antibiotic release from the biomaterial. In this in vitro study, hydroxyapatite biomaterials with antibiotics and biodegradable polymers were tested for their ability to reduce bacteria adhesion and biofilm development. This study examined the antibacterial efficiency of hydroxyapatite biomaterials with antibiotics and biodegradable polymers against Staphylococcus epidermidis and Pseudomonas aeruginosa. The study found that hydroxyapatite biomaterials with antibiotics and biodegradable polymers show longer antibacterial properties than hydroxyapatite biomaterials with antibiotics against both bacterial cultures. Therefore, the results of this study demonstrated that biomaterials that are coated with biodegradable polymers release antibiotics from biomaterial samples for a longer period of time and may be useful for reducing bacterial adhesion on orthopedic implants.

Low-temperature-processed biocompatible Ag-HAp nanoparticles with anti-biofilm efficacy for tissue engineering applications

Biomaterial-associated infections are the major cause of implant failure and can also develop many years after implantation. Recent advances in nanotechnology and the development of new nano-materials have led to the design of anti-biofilm coatings on implant surfaces. In this study, the inhibition of biofilm formation on silver-doped hydroxyapatite (Ag-HAp) nanoparticles-coated glass slides is reported. Ag-HAp was synthesized using low-temperature-modified sol–gel method. The release of silver ions from the Ag-HAp reduced the adhesion and prevented formation of biofilms of Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa when studied over a 10-day period. These coatings released an initial high amount of silver ions followed by a slow and gradual release facilitating sustained anti-biofilm activity. This initial high release of silver is beneficial for reducing bacterial adhesion which is the first step in the development of a biofilm. The biocompatibility of silver-doped hydroxyapatite coatings has also been confirmed. As success or failure of an implant depends on the balance between host tissue integration and bacterial colonization, the competitive growth between the mammalian and bacterial cells by inoculating E. coli into actively growing MG63 osteosarcoma cells on Ag-HAp coatings has been investigated. This could mimic the peri-operative contamination model. Ag-HAp-coated slides exhibit anti-biofilm activity over a period of 10 days, while supporting the growth of osteosarcoma cells (MG63). Co-culturing of MG63 cells with bacteria showed healthy growth of MG63 cells and no growth of bacteria. HAp control did not support growth of MG63 cells in the presence of bacteria.

Bio-Inspired Coating Strategies for the Immobilization of Polymyxins to Generate Contact-Killing Surfaces.

Microbial colonization of indwelling devices remains a major concern in modern healthcare. Developing approaches to prevent biomaterial-associated infections (BAI) is, therefore, in great demand. This study aimed to immobilize two antimicrobial peptides (polymyxins B and E) onto polydimethylsiloxane (PDMS) using two polydopamine (pDA)-based approaches: the conventional two-step method involving the deposition of a pDA layer to which biomolecules are immobilized, and a one-step method where peptides are dissolved together with dopamine before its polymerization. Surface characterization confirms the immobilization of polymyxins onto PDMS at a non-toxic concentration. Immobilization of polymyxins using a one-step pDA-based approach is able to prevent Pseudomonas aeruginosa adhesion and kill a significant fraction of the adherent ones. Living cells adhered to these modified surfaces exhibit the same susceptibility pattern as cells adhered to unmodified surfaces, highlighting no resistance development. Results suggest that polymyxins immobilization holds a great potential as an additional antimicrobial functionality in the design of biomaterials.

High Antimicrobial Activity and Low Human Cell Cytotoxicity of Core-Shell Magnetic Nanoparticles Functionalized with an Antimicrobial Peptide.

Superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with antimicrobial agents are promising infection-targeted therapeutic platforms when coupled with external magnetic stimuli. These antimicrobial nanoparticles (NPs) may offer advantages in fighting intracellular pathogens as well as biomaterial-associated infections. This requires the development of NPs with high antimicrobial activity without interfering with the biology of mammalian cells. Here, we report the preparation of biocompatible antimicrobial SPION@gold core-shell NPs based on covalent immobilization of the antimicrobial peptide (AMP) cecropin melittin (CM) (the conjugate is named AMP-NP). The minimal inhibitory concentration (MIC) of the AMP-NP for Escherichia coli was 0.4 μg/mL, 10-times lower than the MIC of soluble CM. The antimicrobial activity of CM depends on the length of the spacer between the CM and the NP. AMP-NPs are taken up by endothelial (between 60 and 170 pg of NPs per cell) and macrophage (between 18 and 36 pg of NPs per cell) cells and accumulate preferentially in endolysosomes. These NPs have no significant cytotoxic and pro-inflammatory activities for concentrations up to 200 μg/mL (at least 100 times higher than the MIC of soluble CM). Our results in membrane models suggest that the selectivity of AMP-NPs for bacteria and not eukaryotic membranes is due to their membrane compositions. The AMP-NPs developed here open new opportunities for infection-site targeting.

Antibacterial activity of a new broad-spectrum antibiotic covalently bound to titanium surfaces.

Biofilm-associated infections, particularly those caused by Staphylococcus aureus, are a major cause of implant failure. Covalent coupling of broad-spectrum antimicrobials to implants is a promising approach to reduce the risk of infections. In this study, we developed titanium substrates on which the recently discovered antibacterial agent SPI031, a N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol, was covalently linked (SPI031-Ti). We found that SPI031-Ti substrates prevent biofilm formation of S. aureus and Pseudomonas aeruginosa in vitro, as quantified by plate counting and fluorescence microscopy. To test the effectiveness of SPI031-Ti substrates in vivo, we used an adapted in vivo biomaterial-associated infection model in mice in which SPI031-Ti substrates were implanted subcutaneously and subsequently inoculated with S. aureus. Using this model, we found a significant reduction in biofilm formation (up to 98%) on SPI031-Ti substrates compared to control substrates. Finally, we demonstrated that the functionalization of the titanium surfaces with SPI031 did not influence the adhesion and proliferation of human cells important for osseointegration and bone repair. In conclusion, these data demonstrate the clinical potential of SPI031 to be used as an antibacterial coating for implants, thereby reducing the incidence of implant-associated infections. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2191-2198, 2016.

Nano-thick calcium oxide armed titanium: boosts bone cells against methicillin-resistant Staphylococcus aureus.

Since the use of systemic antibiotics for preventing acute biomaterial-associated infections (BAIs) may build up bacterial resistance and result in huge medical costs and unpredictable mortality, new precaution strategies are required. Here, it demonstrated that titanium armed with a nano-thick calcium oxide layer was effective on averting methicillin-resistant Staphylococcus aureus (MRSA) infections in rabbits. The calcium oxide layer was constructed by, firstly, injecting of metallic calcium into titanium via a plasma immersion ion implantation process, and then transforming the outer most surface into oxide by exposing to the atmosphere. Although the calcium oxide armed titanium had a relative low reduction rate (~74%) in growth of MRSA in vitro, it could markedly promote the osteogenic differentiation of bone marrow stem cells (BMSCs), restore local bone integration against the challenge of MRSA, and decrease the incidence of MRSA infection with a rate of 100% (compared to the titanium control). This study demonstrated for the first time that calcium, as one of the major elements in a human body, could be engineered to avert MRSA infections, which is promising as a safe precaution of disinfection for implantable biomedical devices.

A novel soft tissue model for biomaterial-associated infection and inflammation

Event Abstract Back to Event A novel soft tissue model for biomaterial-associated infection and inflammation Sara Svensson1, 2*, Margarita Trobos1, 2*, Maria Hoffman1, 2*, Birgitta Norlindh1, 2*, Sarunas Petronis2, 3*, Jukka Lausmaa2, 3*, Felicia Suska1, 2 and Peter Thomsen1, 2* 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 SP Technical Research Institute of Sweden, Chemistry, Materials and Surfaces - Medical Device Technology, Sweden Introduction: Biomaterial-associated infection is an important complication of medical devices that often requires removal of the implant for successful healing. The mechanisms of bacterial persistence and how the host immune cells are affected by the presence of a colonized biomaterial are incompletely understood. The exploration of novel infection control strategies would be enabled by the development of experimental models allowing the quantitative determination of host-bacteria interactions in vivo. This study describes a soft tissue model for the investigation of bacteriological, morphological and molecular events taking place on the implant surface and in the surrounding exudate and tissue[1]. Materials and Methods: Smooth and nanostructured Ti disks with or without noble metal chemistry (Ag, Au, Pd) and sham sites were inoculated with a dose of 106 CFU Staphylococcus epidermidis (ATCC 35984) subcutaneously on the back of rats. Implant and sham sites were analysed after 4 h, 24 h and 72 h with respect to the number of viable bacteria and the number, viability and gene expression (TNF-α, IL-6, IL-8, IL-10, TLR2, TLR4, elastase) of host cells. The tissue response and the location of S. epidermidis in the interface tissue, were evaluated by histology and FISH, respectively. Non-infected rats were controls. Data was statistically evaluated in PASW/Statistics 18.0 and different tests were applied depending on the type of comparison (n=7-8). The study was approved by the local ethical committee for laboratory animals (Dnr 254/11). Results and Discussion: The results revealed distinct differences between infected and control sites, e.g. in the number, type and viability of host cells surrounding the implant. Host cells at infected sites showed significantly higher gene expression activity of genes related to inflammation, bacterial recognition and tissue degradation than control sites. In the tissue, bacterial cells were primarily located in close proximity to the implant, both extra- and intracellularly (Fig 1). Cytospin preparations of the exudate showed that both mono- and polymorphonuclear cells contained bacterial cells within the cytoplasm (Fig 2). Comparisons between materials revealed elevated cell recruitment, higher neutrophil presence and higher pro-inflammatory gene expression in host cells surrounding noble metal coated Ti, which was in addition associated with a lower number of viable bacteria at 24 h (Fig 3). Interestingly, all implant surfaces harboured less bacteria (ten fold) than the surrounding exudate at all time points. In addition, S. epidermidis still persisted at both material and sham sites after 72 h, indicating a need for longer-term studies. Conclusion: This model allows detailed analysis of early events in inflammation and infection associated to biomaterials in vivo, leading to insights into host defence mechanisms in biomaterial-associated infections. BIOMATCELL Vinn Excellence Center of Biomaterials and Cell Therapy; Swedish Research Council (grant (K2015-52X-09495-28-4); Hjalmar Svensson Research Foundation; IngaBritt and Arne Lundberg Foundation; Felix Neubergh Foundation

Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants

Event Abstract Back to Event Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants Magdalena Zaborowska1, 2, Rickard Brånemark2, 3, 4, Joakim Strömberg3, Peter Thomsen1, 2 and Margarita Trobos1, 2 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, Department of Orthopaedics, Sweden 4 Department of Orthopaedics, University of California, International Center for Osseointegration Research Education and Surgery (iCORES), United States Introduction: Biomaterial-associated infections (BAI) are among the main reasons for implant failure, often leading to long-term antimicrobial treatment and implant removal. BAI are mainly caused by biofilm producing bacteria with increased antimicrobial resistance. Proper and rapid diagnosis is important for successful treatment. Sonication is an alternative method for the diagnosis of prosthetic infections with higher sensitivity than standard tissue cultures[1]. The aims of the study were to: i) identify and quantify bacteria causing osteomyelitis associated with osseointegrated percutaneous amputation prostheses by sonication of explanted implants; ii) characterise the isolated strains by their biofilm production abilities and antimicrobial susceptibility. In parallel, the diagnostic laboratory at the hospital performed standard culturing techniques of bone tissue and blood marrow samples. Materials and Methods: A bone-anchored part (fixture) of a prosthesis was extracted due to suspicion of infection according to clinical evaluation. The retrieved fixture was placed in sterile saline, sonicated at 47 KHz for 5 min and vortexed for 1 min. The implant was removed, the remaining solution was centrifuged, pellet was re-suspended in saline and quantitative cultures were made on general purpose agar (blood agar for aerobic and brucella agar for anaerobic cultures) and selective agar plates (staphylococci agar, enterococci agar, streptococci agar, chromogenic orientation agar and pseudomonas agar). Crystal violet (CV) staining and Congo Red (CR) agar plate test was performed to evaluate the biofilm production. Identification of the staphylococcal strains was performed using API Staph. The Calgary Biofilm Device and a custom-made antimicrobial susceptibility plate were used to determine minimum biofilm eradication concentrations (MBEC) and minimum inhibitory concentrations (MIC) to 10 antimicrobial agents. Results and Discussion: We report the first results of an ongoing prospective study where the method has been tested real-time. In the first patient, Enterococcus faecalis (5x104 CFU/implant) and Escherichia coli (102 CFU/implant) were detected after sonication. These strains were both biofilm-producing strains according to CV and CR tests. Biofilms showed increased tolerance to the antimicrobials compared with the standard planktonic MIC values (Table 1). Escherichia coli was the only species detected by standard tissue cultures; in contrast, by the use of sonication, Enterococcus faecalis was further identified indicating the possibility of a polymicrobial infection with potential implications for the choice of the optimal treatment strategy for the patient. Conclusions: An increased detection of the potential causative agents by sonication of retrieved implants may improve the current diagnostics of BAI. In addition, determination of MBEC for clinically relevant antimicrobial agents has the potential to optimize the treatment choices for patients. The BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Västra Götaland Region, the Swedish Research Council (K2015-52X-09495-28-4), an LUA/ALF Research Grant (ALFGBG-138721), the IngaBritt and Arne Lundberg Foundation, the Wilhelm and Martina Lundgren Foundation, the Handlanden Hjalmar Svensson Foundation, Doctor Felix Neubergh Foundation and the Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative launched by the Swedish Government

The development of supramolecular antimicrobial bioactive materials

Event Abstract Back to Event The development of supramolecular antimicrobial bioactive materials Sabrina Zaccaria1, 2, Peter-Paul K. Fransen1, 2, Serge H M Söntjens3, Anton W. Bosman4, Tristan Mes4 and Patricia Y. Dankers1, 2 1 Eindhoven University of Technology, Department of Biomedical Engineering, Laboratory of Chemical Biology, Netherlands 2 Institute for Complex Molecular Systems, Netherlands 3 SyMO-Chem B.V., Netherlands 4 SupraPolix B.V., Netherlands Introduction: Preventing biomaterial associated infections represents one of the main challenges for a new generation of biomaterials, which can significantly improve the quality of life. One strategy to achieve this goal is the coating of medical devices and implants with antimicrobial agents. Among these, Antimicrobial Peptides (AMPs) are currently the most promising, mainly because of their broad spectrum of action and low risk to promote bacterial resistance[1]. In this context, our research aims to establish a new methodology for the incorporation of AMPs into biodegradable implantable polymers based on supramolecular interactions. A modular approach based on supramolecular building blocks enables to easily prepare instructive, biodegradable scaffolds by simply mixing-and-matching of short prepolymers and peptides functionalized with hydrogen-bonding ureido-pyrimidinone (UPy) moieties[2]. In addition, different bioactives can easily be added, enabling to prepare multifunctional biomaterial and to fine tune them according to specific clinical needs. Indeed, tissue engineering and regenerative medicine often require tissue integration; this can be achieved by enhancing autologous cell adhesion on the biomaterial. Here we report on the development of a cell-adhesive antimicrobial supramolecular biomaterial with improved in-situ tissue regenerative capacity for cardiovascular, renal and urological applications, obtained by simply mixing UPy-polymers with various UPy-functionalized peptides promoting both eukaryotic cell adhesion (e.g. RGD)[3] and antimicrobial activity (Figure 1). Materials and Methods: All the peptides were synthesized via Fmoc-based manual solid phase peptide synthesis (SPPS). A carboxylic acid UPy-synthon (UPy-COOH) was coupled to the N-terminus of the peptide using HATU. Purification of the UPy-peptides was carried out through preparative HPLC. Results and Discussion: A small library of UPy-functionalized peptides is designed and synthesized. UPy-functionalized RGD peptides were easily prepared by coupling a UPy-COOH synthon to the protected peptides, while these were still anchored to the resin on which they were synthesized. Antimicrobial peptide sequences were chosen from literature[4]-[6]. Different conditions for Fmoc solid phase strategy have been explored to succeed in the synthesis of the AMPs. The best results in terms of yields and purity were obtained using Sieber amide resin, OxymaPure/DIPCDI as coupling agent and adding a surfactant (Triton-X) to improve the swelling of the resin. Attempts to couple the UPy-COOH synthon to AMPs using the same solid-phase procedure developed for the RGD peptides failed to give the desired products. An in solution strategy for coupling AMPs to the UPy-synthon was therefore developed: AMPs synthesized on Sieber resin were cleaved under mild acidic conditions and then coupled to the UPy-COOH synthon in solution using HATU as coupling agent. Conclusions: A library of UPy-peptides with either cell-adhesive or antimicrobial properties was successfully prepared. These peptides are going to be used in combination with UPy-polymers to prepare supramolecular biomaterials able to induce tissue formation while preventing infections. Acknowledgements: This research has received funding from the Union‘s Seventh Framework Programme (FP/2007-2013) under Grant Agreement number 310389 (BIP-UPy).

Site-specific gene expression analysis of implant-near cells in a soft tissue infection model - application of laser microdissection to study biomaterial-associated infection and inflammation

Event Abstract Back to Event Site-specific gene expression analysis of implant-near cells in a soft tissue infection model - application of laser microdissection to study biomaterial-associated infection and inflammation Sara Svensson1, 2, Margarita Trobos1, 2, Omar Omar1, 2 and Peter Thomsen1, 2 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden Introduction: Medical implants are often used with high success rates. However, some implants fail due to e.g. lack of integration or infection. Resolving the biological events at the implant-tissue interface is essential for proper understanding of the mechanisms behind the success or failure. Laser microdissection (LMD) provides possibilities to harvest specific tissue sites or cells around the implant for subsequent downstream analysis. In the present study, we have analysed the gene expression of cells at increasing distances from titanium (Ti) implants – with or without the presence of Staphylococcus epidermidis. Materials and Methods: Ti disks ± 106 CFU dose of S. epidermidis (ATCC 35984) were implanted subcutaneously in rats. After 3 days, implants were removed and the surrounding tissue was embedded in paraffin. Sections were stained and subjected to LMD (PALM Microbeam microscope, Carl Zeiss MicroImaging). Three tissue zones were dissected at increasing distance from the implant (Zone 1 < Zone 2 < Zone 3) and processed for subsequent qPCR analysis (Fig. 1). Relative gene expression was analysed for TNF-α, IL-6, IL-8, MCP-1, IL-8R, TLR2 and MMP9, using 18S as reference gene. Five full paraffin sections served as an overall tissue reference. The Mann-Whitney U Test was used for statistical analysis (n=4-8). The study was approved by the local ethical committee for laboratory animals (Dnr 254/11). Results and Discussion: Previous results from this soft tissue infection model have shown large differences in the recruited cell number, cell type and gene expression in the implant-surrounding exudate, depending on bacterial presence[1]. The present results on the interface tissue extend the previous findings confirming higher expression levels of inflammatory cytokines and chemokines at infected sites compared with control (Fig. 2). Moreover, a spatial difference in the molecular activity was demonstrated. The gene expression was generally more pronounced at the implant-adjacent interface zone compared with zones at further distance from the implant. Furthermore, high expression of IL-6 and IL-8 was also detected in the distant zones in the infected sites, suggesting a possible chemotaxis gradient. Analysis of the overall “black box” tissue response consistently showed higher gene expression in the infected sites, without providing any details of the spatial distribution of the cellular activity triggered by the presence of bacteria. Conclusion: The results show spatial differences in host cell gene expression depending on the distance from an implant surface. Furthermore, the study demonstrates the use of LMD as a powerful tool for molecular dissection of the implant-tissue interface during normal healing or in association with infection. BIOMATCELL Vinn Excellence Center of Biomaterials and Cell Therapy; Swedish Research Council (grant (K2015-52X-09495-28-4); Hjalmar Svensson Research Foundation; Birgitta Norlindh

Fabrication of antibacterial and hydrophilic electroless Ni–B coating on 316L stainless steel

Biomaterial-associated bacterial infection is one of the most common complications with medical vehicles and implants made of stainless steel. A surface coating treatment like electroless Ni–B deposition, a new candidate to be used in a broad range of engineering applications owing to many advantages such as low cost, thickness uniformity, good wear resistance, may improve the antibacterial activity and physical properties of biomedical devices made of stainless steel. In this study, the antibacterial property of the electroless Ni–B film coated on AISI 316L (UNS S31603) stainless steel is basically investigated. Inhibition halo diameter measurement after incubation at 37 °C and 24 h demonstrates the existence of antimicrobial activity of the electroless Ni–B coating deposited on 316L stainless steel over the Escherichia coli test bacteria. The results of X-ray diffraction, scanning electron microscopy, atomic force microscopy and microhardness measurement studies confirms that the coating deposited on the substrate has an uniform amorphous and a harder structure. Besides, the wettability property of the uncoated substrate and the coating was measured as the contact angle of water. The water contact angle reduced about from 97.7 to 69.25°.

Covalent immobilization of antimicrobial agents on titanium prevents Staphylococcus aureus and Candida albicans colonization and biofilm formation.

Biofilm-associated implant infections represent a serious public health problem. Covalent immobilization of antimicrobial agents on titanium (Ti), thereby inhibiting biofilm formation of microbial pathogens, is a solution to this problem. Vancomycin (VAN) and caspofungin (CAS) were covalently bound on Ti substrates using an improved processing technique adapted to large-scale coating of implants. Resistance of the VAN-coated Ti (VAN-Ti) and CAS-coated Ti (CAS-Ti) substrates against in vitro biofilm formation of the bacterium Staphylococcus aureus and the fungal pathogen Candida albicans was determined by plate counting and visualized by confocal laser scanning microscopy. The efficacy of the coated Ti substrates was also tested in vivo using an adapted biomaterial-associated murine infection model in which control-Ti, VAN-Ti or CAS-Ti substrates were implanted subcutaneously and subsequently challenged with the respective pathogens. The osseointegration potential of VAN-Ti and CAS-Ti was examined in vitro using human bone marrow-derived stromal cells, and for VAN-Ti also in a rat osseointegration model. In vitro biofilm formation of S. aureus and C. albicans on VAN-Ti and CAS-Ti substrates, respectively, was significantly reduced compared with biofilm formation on control-Ti. In vivo, we observed over 99.9% reduction in biofilm formation of S. aureus on VAN-Ti substrates and 89% reduction in biofilm formation of C. albicans on CAS-Ti substrates, compared with control-Ti substrates. The coated substrates supported osseointegration in vitro and in vivo. These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

Protein adsorption, platelet adhesion, and bacterial adhesion to polyethylene-glycol-textured polyurethane biomaterial surfaces.

Traditional strategies for surface modification to enhance the biocompatibility of biomaterials often focus on a single route utilizing either chemical or physical approaches. This study combines the chemical and physical treatments as applied to poly(urethane urea) (PUU) biomaterials to enhance biocompatibility at the interface for inhibiting platelet-related thrombosis or bacterial adhesion-induced microbial infections. PUU films were first textured with submicron patterns by a soft lithography two-stage replication process, and then were grafted with polyethylene glycol (PEG). A series of biological response experiments including protein adsorption, platelet adhesion/activation, and bacterial adhesion/biofilm formation showed that PEG-grafted submicron textured biomaterial surfaces were resistant to protein adsorption, and greatly increased the efficiency in reducing both platelet adhesion/activation and bacterial adhesion/biofilm formation due to the additive effects of physical topography and grafted PEG. Results suggest that a combination of chemical modification and surface texturing will be more efficient in preventing biomaterial-associated thrombosis and infection of biomaterials. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 668-678, 2017.

Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145

The scarcity of current antibiotic-based strategies to prevent biomaterial-associated infections (BAI) and their risk of resistance development prompted us to develop a novel antimicrobial implant-coating to prevent Staphylococcus aureus-induced BAI. We incorporated the antimicrobial peptide OP-145 into a Polymer-Lipid Encapsulation MatriX (PLEX)-coating to obtain high peptide levels for prolonged periods at the implant-tissue interphase. We first confirmed that OP-145 was highly effective in killing S. aureus and inhibiting biofilm formation in vitro. OP-145 injected along S. aureus-inoculated implants in mice significantly reduced the number of culture-positive implants. OP-145 was released from the PLEX coating in a controlled zero-order kinetic rate after an initial 55%-burst release and displayed bactericidal activity in vitro. In a rabbit intramedullary nail-related infection model, 67% of rabbits with PLEX-OP-145-coated nails had culture-negative nails after 28days compared to 29% of rabbits with uncoated nails. In rabbits with PLEX-OP-145-coated nails, bone and soft tissue samples were culture-negative in 67% and 80%, respectively, whereas all bone samples and 71% of the soft tissue samples of rabbits with uncoated nails were infected. Together, PLEX-OP-145 coatings, of which both compounds have already been found safe in man, can prevent implant colonization and S. aureus-induced BAIs.

Antibacterial coating of implants in orthopaedics and trauma: a classification proposal in an evolving panorama.

Implanted biomaterials play a key role in current success of orthopedic and trauma surgery. However, implant-related infections remain among the leading reasons for failure with high economical and social associated costs. According to the current knowledge, probably the most critical pathogenic event in the development of implant-related infection is biofilm formation, which starts immediately after bacterial adhesion on an implant and effectively protects the microorganisms from the immune system and systemic antibiotics. A rationale, modern prevention of biomaterial-associated infections should then specifically focus on inhibition of both bacterial adhesion and biofilm formation. Nonetheless, currently available prophylactic measures, although partially effective in reducing surgical site infections, are not based on the pathogenesis of biofilm-related infections and unacceptable high rates of septic complications, especially in high-risk patients and procedures, are still reported.In the last decade, several studies have investigated the ability of implant surface modifications to minimize bacterial adhesion, inhibit biofilm formation, and provide effective bacterial killing to protect implanted biomaterials, even if there still is a great discrepancy between proposed and clinically implemented strategies and a lack of a common language to evaluate them.To move a step forward towards a more systematic approach in this promising but complicated field, here we provide a detailed overview and an original classification of the various technologies under study or already in the market. We may distinguish the following: 1. Passive surface finishing/modification (PSM): passive coatings that do not release bactericidal agents to the surrounding tissues, but are aimed at preventing or reducing bacterial adhesion through surface chemistry and/or structure modifications; 2. Active surface finishing/modification (ASM): active coatings that feature pharmacologically active pre-incorporated bactericidal agents; and 3. Local carriers or coatings (LCC): local antibacterial carriers or coatings, biodegradable or not, applied at the time of the surgical procedure, immediately prior or at the same time of the implant and around it. Classifying different technologies may be useful in order to better compare different solutions, to improve the design of validation tests and, hopefully, to improve and speed up the regulatory process in this rapidly evolving field.

The role of programmed death ligand 1 pathway in persistent biomaterial-associated infections.

Staphylococcus epidermidis is commonly involved in biomaterial-associated infections. Bacterial small colony variants (SCV) seem to be well adapted to persist intracellularly in professional phagocytes evading the host immune response. We studied the expression of PD-L1/L2 on macrophages infected with clinical isolates of S. epidermidis SCV and their parent wild type (WT) strains. The cytokine pattern which is triggered by the examined strains was also analysed. In the study, we infected macrophages with S. epidermidis WT and SCV strains. Persistence and release from macrophages were monitored via lysostaphin protection assays. Moreover, the effect of IFN-γ pre-treatment on bacterial internalisation was investigated. Expression of PD-L1/L2 molecules was analysed with the use of FACS. Inflammatory reaction was measured by IL-10, TNF-α ELISAs, and transcriptional induction of TNF-α. Our study revealed that clinical SCV isolates were able to persist and survive in macrophages for at least 3 days with a low cytotoxic effect and a reduced proinflammatory response as compared to WT strains. Bacteria upregulated PD-L1/L2 expression on macrophages as compared to non-stimulated cells. The results demonstrated that the ability of S. epidermidis SCVs to induce elevated levels of anti-inflammatory cytokine, IL-10, and reduced transcriptional induction of TNF-α, together with expression of PD-L1 on macrophages and the ability to persist intracellularly without damaging the host cell could be the key factor contributing to chronicity of SCV infections.