Reduced Cell Adhesion on LightPLAS-Coated Implant Surfaces in a Three-Dimensional Bioreactor System.

Most implants used in trauma surgery are made of steel and remain inside the body only temporarily. The strong tissue interaction of such implants sometimes creates problems with their explantation. Modified implant surfaces, which decrease tissue attachment, might allow an easier removal and therefore a better outcome. Such a modification must retain the implant function, and needs to be biocompatible and cost-effective. Here, we used a novel VUV-light (Vacuum-Ultraviolett)-based coating technology (LightPLAS) to generate coated stainless-steel plates. The tested LightPLAS coating only had an average thickness of around 335 nm, making it unlikely to interfere with implant function. The coated plates showed good biocompatibility according to ISO 10993-5 and ISO 10993-12, and reduced cell adhesion after four different time points in a 2D cell culture system with osteoblast-like MG-63 cells. Furthermore, we could show decreased cell adhesion in our 3D cell culture system, which mimics the fluid flow above the implant materials as commonly present in the in vivo environment. This new method of surface coating could offer extended options to design implant surfaces for trauma surgery to reduce cell adhesion and implant ingrowth. This may allow for a faster removal time, resulting in shorter overall operation times, thereby reducing costs and complication rates and increasing patient wellbeing.

Impact of Laser Structuring on Medical-Grade Titanium: Surface Characterization and In Vitro Evaluation of Osteoblast Attachment.

Improved implant osteointegration offers meaningful potential for orthopedic, spinal, and dental implants. In this study, a laser treatment was used for the structuring of a titanium alloy (Ti6Al4V) surface combined with a titanium dioxide coating, whereby a porous surface was created. The objective was to characterize the pore structure shape, treatment-related metallographic changes, cytocompatibility, and attachment of osteoblast-like cells (MG-63). The treatment generated specific bottleneck pore shapes, offering the potential for the interlocking of osteoblasts within undercuts in the implant surface. The pore dimensions were a bottleneck diameter of 27 µm (SD: 4 µm), an inner pore width of 78 µm (SD: 6 µm), and a pore depth of 129 µm (SD: 8 µm). The introduced energy of the laser changed the metallic structure of the alloy within the heat-affected region (approximately 66 µm) without any indication of a micro cracking formation. The phase of the alloy (microcrystalline alpha + beta) was changed to a martensite alpha phase in the surface region and an alpha + beta phase in the transition region between the pores. The MG-63 cells adhered to the structured titanium surface within 30 min and grew with numerous filopodia over and into the pores over the following days. Cell viability was improved on the structured surface compared to pure titanium, indicating good cytocompatibility. In particular, the demonstrated affinity of MG-63 cells to grow into the pores offers the potential to provide significantly improved implant fixation in further in vivo studies.

Burst Release of Antibiotics Combined with Long-Term Release of Silver Targeting Implant-Associated Infections: Design, Characterization and in vitro Evaluation of Novel Implant Hybrid Surface.

Implant-associated infections represent a serious risk in human medicine and can lead to complications, revisions and in worst cases, amputations. To target these risks, the objective was to design a hybrid implant surface that allows a local burst release of antibiotics combined with long-term antimicrobial activity based on silver. The efficacy should be generated with simultaneous in vitro cytocompatibility. The investigations were performed on titanium K-wires and plates and gentamicin was selected as an illustrative antibiotic. A gentamicin depot (max 553 µg/cm2) was created on the surface using laser structuring. The antibiotic was released within 15 min in phosphate buffered saline (PBS) or agar medium. Metallic silver particles (4 µg/cm2) in a titanium dioxide layer were deposited using plasma vapor deposition (PVD). About 16% of the silver was released within 28 days in the agar medium. The local efficacy of the incorporated silver was demonstrated in a direct contact assay with a reduction of more than 99.99% (Escherichia coli). The local efficacy of the hybrid surface was confirmed in a zone of inhibition (ZOI) assay using Staphylococcus cohnii. The biocompatibility of the hybrid surface was proven using fibroblasts and osteoblasts as cell systems. The hybrid surface design seems to be promising as treatment of implant-associated infections, considering the achieved amount and release behavior of the active ingredients (gentamicin, silver). The generated in vitro results (efficacy, biocompatibility) proofed the concept. Further in vivo studies will be necessary translate the hybrid surface towards clinical applied research.

Functionalization of hydrophobic surfaces with antimicrobial peptides immobilized on a bio-interfactant layer.

The design of functionalized polymer surfaces using bioactive compounds has grown rapidly over the past decade within many industries including biomedical, textile, microelectronics, bioprocessing and food packaging sectors. Polymer surfaces such as polystyrene (PS) must be treated using surface activation processes prior to the attachment of bioactive compounds. In this study, a new peptide immobilization strategy onto hydrocarbonaceus polymer surfaces is presented. A bio-interfactant layer made up of a tailored combination of laccase from trametes versicolor enzyme and maltodextrin is applied to immobilize peptides. Using this strategy, immobilization of the bio-inspired peptide KLWWMIRRWG-bromophenylalanine-3,4-dihydroxyphenylalanine-G and KLWWMIRRWG-bromophenylalanine-G on polystyrene (PS) was achieved. The interacting laccase layers allows to immobilize antimicrobial peptides avoiding the chemical modification of the peptide with a spacer and providing some freedom that facilitates different orientations. These are not strongly dominated by the substrate as it is the case on hydrophobic surfaces; maintaining the antimicrobial activity. Films exhibited depletion efficiency with respect to the growth of Escherichia coli bacteria and did not show cytotoxicity for fibroblast L929. This environmentally friendly antimicrobial surface treatment is both simple and fast, and employs aqueous solutions. Furthermore, the method can be extended to three-dimensional scaffolds as well as rough and patterned substrates.

Development of Ag/AgX (X = Cl, I) nanoparticles toward antimicrobial, UV-protected and self-cleanable viscose fibers.

In situ synthesis of Ag/AgX nanoparticles (NPs) onto viscose fibers adds new functionalities and broadens their applications. In this study, Ag/AgX (X = Cl, I) NPs were in situ synthesized onto viscose fibers to impart brilliant colors, UV-protection, antimicrobial, self-cleaning, and photocatalytic properties. The AgX NPs were deposited on the fibers by ultrasonic irradiation, while Ag-NPs were formed by photoreduction of excess Ag+ ions under UV irradiation. The Ag/AgX NPs-loaded onto viscose fibers endowed with pale yellow for Ag/AgI and pale purple/violet for Ag/AgCl. The colored viscose fibers showed excellent antimicrobial activity against Escherichia coli (gram-negative), Staphylococcus aureus (Gram positive), and Candida Albican. The Ag/AgX/viscose fiber also showed excellent photocatalytic and self-cleaning activity toward degradation of methylene blue.

Towards multifunctional cellulosic fabric: UV photo-reduction and in-situ synthesis of silver nanoparticles into cellulose fabrics.

Herein, the highly multifunctional cotton fabric surfaces were designed with excellent coloration, UV-protection function, and antimicrobial activity. These multifunctional functions were developed by in-situ synthesis of silver nanoparticles (Ag NPs) into the cotton fabric surface using a simple green one-pot "UV-reduction" method. Cotton fabrics were pretreated with non-anionic detergent, immersed into alcoholic silver nitrate solution (concentration ranging from 100 to 500ppm), squeezed to remove excess solution and then exposed to UV-irradiation (range 320-400nm) for 1h. The influence UV-irradiation on the thermal, chemical, optical and biological properties of the cotton fabric surface was discussed in details. The UV-irradiation promotes reducing of Ag+ ions and the cotton fabrics act as seed medium for Ag NPs formation by "heterogeneous nucleation". Increasing Ag+ concentration (from 100 to 500ppm) results in Ag NPs of particle size (distribution) of 50-100nm. Interestingly, the Ag NPs exhibited different localized surface Plasmon resonance properties causing a coloration of the cotton fabrics with different color shades ranging from bright to dark brown with excellent color fastness properties. The treated cotton fabrics also show high protecting functions against UV-transmission (reduction of 65%) and Escherichia coli growth (99%). The side-effects of the UV-reduction process are further investigated.

Evolution toward small molecule inhibitor resistance affects native enzyme function and stability, generating acarbose-insensitive cyclodextrin glucanotransferase variants.

Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC(50) values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants.