Annabel Braem

Staphylococcal biofilm growth on smooth and porous titanium coatings for biomedical applications

Implant-related infections are a serious complication in prosthetic surgery, substantially jeopardizing implant fixation. As porous coatings for improved osseointegration typically present an increased surface roughness, their resulting large surface area (sometimes increasing with over 700% compared to an ideal plane) renders the implant extremely susceptible to bacterial colonization and subsequent biofilm formation. Therefore, there is particular interest in orthopaedic implantology to engineer surfaces that combine both the ability to improve osseointegration and at the same time reduce the infection risk. As part of this orthopaedic coating development, the interest of in vitro studies on the interaction between implant surfaces and bacteria/biofilms is growing. In this study, the in vitro staphylococcal adhesion and biofilm formation on newly developed porous pure Ti coatings with 50% porosity and pore sizes up to 50 μm is compared to various dense and porous Ti or Ti-6Al-4V reference surfaces. Multiple linear regression analysis indicates that surface roughness and hydrophobicity are the main determinants for bacterial adherence. Accordingly, the novel coatings display a significant reduction of up to five times less bacterial surface colonization when compared to a commercial state-of-the-art vacuum plasma sprayed coating. However, the results also show that a further expansion of the porosity with over 15% and/or the pore size up to 150 μm is correlated to a significant increase in the roughness parameters resulting in an ascent of bacterial attachment. Chemically modifying the Ti surface in order to improve its hydrophilicity, while preserving the average roughness, is found to strongly decrease bacteria quantities, indicating the importance of surface functionalization to reduce the infection risk of porous coatings.

Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures.

Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.

Peri- and intra-implant bone response to microporous Ti coatings with surface modification.

Bone growth on and into implants exhibiting substantial surface porosity is a promising strategy in order to improve the long-term stable fixation of bone implants. However, the reliability in clinical applications remains a point of discussion. Most attention has been dedicated to the role of macroporosity, leading to the general consensus of a minimal pore size of 50-100 μm in order to allow bone ingrowth. In this in vivo study, we assessed the feasibility of early bone ingrowth into a predominantly microporous Ti coating with an average thickness of 150 μm and the hypothesis of improving the bone response through surface modification of the porous coating. Implants were placed in the cortical bone of rabbit tibiae for periods of 2 and 4 weeks and evaluated histologically and histomorphometrically using light microscopy and scanning electron microscopy. Bone with osteocytes encased in the mineralized matrix was found throughout the porous Ti coating up to the coating/substrate interface, highlighting that osseointegration of microporosities (<10 μm) was achievable. The bone trabeculae interweaved with the pore struts, establishing a large contact area which might enable an improved load transfer and stronger implant/bone interface. Furthermore, there was a clear interconnection with the surrounding cortical bone, suggesting that mechanical interlocking of the coating in the host bone in the long term is possible. When surface modifications inside the porous structure further reduced the interconnective pore size to the submicrometer level, bone ingrowth was impaired. On the other hand, application of a sol-gel-derived bioactive glass-ceramic coating without altering the pore characteristics was found to significantly improve bone regeneration around the coating, while still supporting bone ingrowth.

Fungal β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia coli in a Polymicrobial E. coli/Candida albicans Biofilm

ABSTRACT In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

Reduction of Biofilm Infection Risks and Promotion of Osteointegration for Optimized Surfaces of Titanium Implants

Titanium-based implants are widely used in modern clinical practice; however, complications associated with implants due to bacterial-induced infections arise frequently, caused mainly by staphylococci, streptococci, Pseudomonas spp. and coliform bacteria. Although increased hydrophilicity of the biomaterial surface is known to be beneficial in minimizing the biofilm, quantitative analyses between the actual implant parameters and bacterial development are scarce. Here, the results of in vitro studies of Staphylococcus aureus and Staphylococcus epidermidis proliferation on uncoated and coated titanium materials with different roughness, porosity, topology, and hydrophilicity are shown. The same materials have been tested in parallel with respect to human osteogenic and endothelial cell adhesion, proliferation, and differentiation. The experimental data processed by meta-analysis are indicating the possibility of decreasing the biofilm formation by 80-90% for flat substrates versus untreated plasma-sprayed porous titanium and by 65-95% for other porous titanium coatings. It is also shown that optimized surfaces would lead to 10-50% enhanced cell proliferation and differentiation versus reference porous titanium coatings. This presents an opportunity to manufacture implants with intrinsic reduced infection risk, yet without the additional use of antibacterial substances.

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS These data demonstrate the clinical potential of covalently bound VAN and CAS on Ti to reduce microbial biofilm formation without jeopardizing osseointegration.

The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms.

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.

Controlled release of chlorhexidine antiseptic from microporous amorphous silica applied in open porosity of an implant surface

Amorphous microporous silica (AMS) serving as a reservoir for controlled release of a bioactive agent was applied in the open porosity of a titanium coating on a Ti-6Al-4V metal substrate. The pores of the AMS emptied by calcination were loaded with chlorhexidine diacetate (CHX) via incipient wetness impregnation with CHX solution, followed by solvent evaporation. Using this CHX loaded AMS system on titanium substrate sustained release of CHX into physiological medium was obtained over a 10 day-period. CHX released from the AMS coating was demonstrated to be effective in killing planktonic cultures of the human pathogens Candida albicans and Staphylococcus epidermidis. This surface modification of titanium bodies with AMS controlled release functionality for a bioactive compound potentially can be applied on dental and orthopaedic implants to abate implant-associated microbial infection.

Novel anti-infective implant substrates: Controlled release of antibiofilm compounds from mesoporous silica-containing macroporous titanium

Bone implants with open porosity enable fast osseointegration, but also present an increased risk of biofilm-associated infections. We design a novel implant material consisting of a mesoporous SiO2 diffusion barrier (pore diameter: 6.4 nm) with controlled drug release functionality integrated in a macroporous Ti load-bearing structure (fully interconnected open porosity: 30%; pore window size: 0.5-2.0 μm). Using an in vitro tool consisting of Ti/SiO2 disks in an insert set-up, through which molecules can diffuse from feed side to release side, a continuous release without initial burst effect of the antibiofilm compound toremifene is sustained for at least 9 days, while release concentrations (up to 17 μM daily) increase with feed concentrations (up to 4mM). Toremifene diffusivity through the SiO2 phase into H2O is estimated around 10(-13)m(2)/s, suggesting configurational diffusion through mesopores. Candida albicans biofilm growth on the toremifene-release side is significantly inhibited, establishing a proof-of-concept for the drug delivery functionality of mesoporous SiO2 incorporated into a high-strength macroporous Ti carrier. Next-generation implants made of this composite material and equipped with an internal reservoir (feed side) can yield long-term controlled release of antibiofilm compounds, effectively treating infections on the implant surface (release side) over a prolonged time.

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.