Andrea Ewald

Antimicrobial titanium/silver PVD coatings on titanium

BackgroundBiofilm formation and deep infection of endoprostheses is a recurrent complication in implant surgery. Post-operative infections may be overcome by adjusting antimicrobial properties of the implant surface prior to implantation. In this work we described the development of an antimicrobial titanium/silver hard coating via the physical vapor deposition (PVD) process.MethodsCoatings with a thickness of approximately 2 μm were deposited on titanium surfaces by simultaneous vaporisation of both metals in an inert argon atmosphere with a silver content of approximately 0.7 – 9% as indicated by energy dispersive X-ray analysis. On these surfaces microorganisms and eukaryotic culture cells were grown.ResultsThe coatings released sufficient silver ions (0.5–2.3 ppb) when immersed in PBS and showed significant antimicrobial potency against Staphylococcus epidermis and Klebsiella pneumoniae strains. At the same time, no cytotoxic effects of the coatings on osteoblast and epithelial cells were found.ConclusionDue to similar mechanical performance when compared to pure titanium, the TiAg coatings should be suitable to provide antimicrobial activity on load-bearing implant surfaces.

Silver-doped calcium phosphate cements with antimicrobial activity.

There is a current need for the localised delivery of antibiotics in order to treat implant-based bacterial infections. Existing treatments use non-resorbable materials such as poly(methyl methacrylate) beads loaded with antibiotics; unfortunately, as they are not resorbable, these beads require secondary surgery for removal. Calcium phosphate cements have considerable potential for the localised delivery of drugs since they can be resorbed to some extent within the body, eliminating the need for a secondary surgical procedure. Therefore, in this study, the efficacy of both hydroxyapatite and brushite cements in the delivery of silver ions has been investigated. The activity of the Ag(+) released from the cements was assessed against the growth of both Staphylococcus aureus and Staphylococcus epidermidis; the brushite cement exhibited excellent antibacterial properties and also showed an increase in compressive strength of over 30%. In this study we have found that with a few changes in Ag(+) concentration it should be possible to produce a fully resorbable bone replacement material that is combined with an antibacterial scaffold with controlled release over a period of time, which is likely to inhibit bacterial infections associated with implantation procedures.

Biofabrication of Cell‐Loaded 3D Spider Silk Constructs

Biofabrication is an emerging and rapidly expanding field of research in which additive manufacturing techniques in combination with cell printing are exploited to generate hierarchical tissue-like structures. Materials that combine printability with cytocompatibility, so called bioinks, are currently the biggest bottleneck. Since recombinant spider silk proteins are non-immunogenic, cytocompatible, and exhibit physical crosslinking, their potential as a new bioink system was evaluated. Cell-loaded spider silk constructs can be printed by robotic dispensing without the need for crosslinking additives or thickeners for mechanical stabilization. Cells are able to adhere and proliferate with good viability over at least one week in such spider silk scaffolds. Introduction of a cell-binding motif to the spider silk protein further enables fine-tuned control over cell-material interactions. Spider silk hydrogels are thus a highly attractive novel bioink for biofabrication.

Photoactivation of CdSe/ZnS Quantum Dots Embedded in Silica Colloids

A study of the influence of the local environment on the light-induced luminescence enhancement of CdSe/ZnS quantum dots (QD) embedded in silica colloids that are dispersed in various solvents is presented. The photoluminescence of the embedded QD is enhanced up to a factor of ten upon photoactivation by ultraviolet or visible light. This enhancement is strongly dependent on the local environment. The thickness-dependent permeability of the silica shell covering the QD controls the influence of the solvent on the QD. If foreign ions are present the activation state is stabilized after termination of the activation, whereas in their absence the process is partially reversible. A new qualitative model for the photoactivation of QD in various environments is developed. It comprises light-induced passivation and subsequent oxidation processes. The embedded QD also retain their fluorescence quantum yield inside living cells. Moreover, they can be activated for many hours in living cells by laser radiation in the visible regime.

Osteoblast response to biomimetically altered titanium surfaces

Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces.

Ready-to-use injectable calcium phosphate bone cement paste as drug carrier

Current developments in calcium phosphate cement (CPC) technology concern the use of ready-to-use injectable cement pastes by dispersing the cement powder in a water-miscible solvent, such that, after injection into the physiological environment, setting of cements occurs by diffusion of water into the cement paste. It has also been demonstrated recently that the combination of a water-immiscible carrier liquid combined with suitable surfactants facilitates a discontinuous liquid exchange in CPC, enabling the cement setting reaction to take place. This paper reports on the use of these novel cement paste formulations as a controlled release system of antibiotics (gentamicin, vancomycin). Cement pastes were applied either as a one-component material, in which the solid drugs were physically dispersed, or as a two-component system, where the drugs were dissolved in an aqueous phase that was homogeneously mixed with the cement paste using a static mixing device during injection. Drug release profiles of both antibiotics from pre-mixed one- and two-component cements were characterized by an initial burst release of ∼7-28%, followed by a typical square root of time release kinetic for vancomycin. Gentamicin release rates also decreased during the first days of the release study, but after ∼1 week, the release rates were more or less constant over a period of several weeks. This anomalous release kinetic was attributed to participation of the sulfate counter ion in the cement setting reaction altering the drug solubility. The drug-loaded cement pastes showed high antimicrobial potency against Staphylococcus aureus in an agar diffusion test regime, while other cement properties such as mechanical performance or phase composition after setting were only marginally affected.

Distribution of emerin during the cell cycle.

Human emerin is a nuclear membrane protein that is lost or altered in patients with Emery-Dreifuss muscular dystrophy (EMD). While the protein is expressed in the majority of human tissues analyzed, the pathology predominates in cardiac and skeletal muscles of patients with EMD. Our results show that emerin can be detected by immunocytochemistry and immunoblotting in the nuclear envelope of all vertebrates studied from man to Xenopus. Immunolocalizations and nuclear envelope extraction experiments confirm that emerin possesses properties characteristic for integral membrane proteins of the inner nuclear membrane. Some nuclear envelope proteins are localized also in annulate lamellae (AL), i.e. cytoplasmic flattened membrane cisternae penetrated by pore complexes. To verify whether emerin is contained in these membrane stacks, we have induced the formation of AL by exposure of rat cells (line RV-SMC) to sublethal doses of the antimitotic drug vinblastine sulfate and found that emerin is present in the nuclear envelope, but is absent from AL. In contrast to the homogeneous distribution of emerin in the nuclear envelope of interphase cells, this protein shows a focal accumulation in the nuclear membranes of late telophase cells. During early reassembly of the nuclear envelope at this mitotic stage emerin colocalizes with lamin A/C but not with lamin B and LAP2 proteins. Confocal laser scanning microscopy after double-labeling experiments with emerin and tubulin shows that emerin is concentrated in areas of the mitotic spindle and in the midbody of mitotic cells suggesting a close interaction of these proteins. Our data suggest that emerin participates in the reorganisation of the nuclear envelope at the end of mitosis.

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

BackgroundThe autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene.ResultsLymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates.ConclusionsFrom our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription.

The effect of Cu(II)-loaded brushite scaffolds on growth and activity of osteoblastic cells

Bone substitute materials such as calcium phosphate cements (CPC) are frequently used as growth factor carriers for the stimulation of osteoblast-formation around an implant. However, biological modification based on delicate protein factors like extracellular matrix proteins or growth factors is subject to a number of shortcomings like the need for storage below room temperature and cost of production. The aim of this study was to investigate ionic modification as an alternative bioinorganic route for implant modification. Although it is known that Cu(II) plays a role in angiogenesis and bone formation, not all involved processes are well understood yet. In this study the in vitro effect of Cu(II) on growth and activity of osteoblastic cells seeded on brushite (CaHPO(4) · 2 H(2) O) scaffolds as well as on glass discs was investigated. The results show that Cu(II) enhances cell activity and proliferation of osteoblastic cells on CPC and furthermore affects the expression of several bone specific proteins such as bone sialo protein or osteocalcin. Therefore, the modification of CPC with Cu(II) may offer a promising alternative to protein based modification to stimulate cellular activity for an improved bone healing.

Strong and tough magnesium wire reinforced phosphate cement composites for load-bearing bone replacement

Calcium phosphate cements are brittle biomaterials of low bending strength. One promising approach to improve their mechanical properties is reinforcement with fibers. State of the art degradable reinforced composites contain fibers made of polymers, resorbable glass or whiskers of calcium minerals. We introduce a new class of composite that is reinforced with degradable magnesium alloy wires. Bending strength and ductility of the composites increased with aspect ratio and volume content of the reinforcements up to a maximal bending strength of 139±41MPa. Hybrid reinforcement with metal and polymer fibers (PLA) further improved the qualitative fracture behavior and gave indication of enhanced strength and ductility. Immersion tests of composites in SBF for seven weeks showed high corrosion stability of ZEK100 wires and slow degradation of the magnesium calcium phosphate cement by struvite dissolution. Finally, in vitro tests with the osteoblast-like cell line MG63 demonstrate cytocompatibility of the composite materials.