Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Anti-inflammatory Surface Coatings Based on Polyelectrolyte Multilayers of Heparin and Polycationic Nanoparticles of Naproxen-Bearing Polymeric Drugs.

Immune response to biomaterials can produce chronic inflammation and fibrosis leading to implant failure, which is related to the surface properties of the biomaterials. This work describes the preparation and characterization of polyelectrolyte multilayer (PEM) coatings that combine the anti-inflammatory activity of heparin as polyanion with the potential release of Naproxen, a nonsteroidal anti-inflammatory drug from polymeric nanoparticles (NP) with cationic surface charge. The polyelectrolyte multilayers were characterized by physical methods to estimate multilayer growth, thickness, zeta potential, and topography. It was found that multilayers with NP had negative zeta potentials and expressed a viscoelastic behavior, while studies of topography showed that nanoparticles formed continuous surface coatings. THP-1-derived macrophages were used to study short-term anti-inflammatory activity (time scale 48 h), showing that PEM that contained heparin reduced cell adhesion and IL1-ß secretion, when compared to those with polystyrenesulfonate, used as alternative polyanion in multilayer formation. On the other hand, the presence of NP in PEM was related to a reduced foreign body giant cell formation after 15 days, when compared to PEM that contained chitosan as alternative polycation, which suggests a long-term anti-inflammatory effect of Naproxen-containing nanoparticles. It was also shown that macrophages were able to take up NP from multilayers, which indicates a release of Naproxen by digestion of NP in the lysosomal compartment. These findings indicate that surface coatings composed of heparin and Naproxen-based NP on implants such as biosensors have the potential to attenuate foreign body reaction after implantation, which may improve the long-term functionality of implants.

The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites.

BACKGROUND: Type VI glandular trichomes represent the most abundant trichome type on leaves and stems of tomato plants and significantly contribute to herbivore resistance, particularly in the wild species. Despite this, their development has been poorly studied so far. The goal of this study is to fill this gap. Using a variety of cell imaging techniques, a detailed record of the anatomy and developmental stages of type VI trichomes in the cultivated tomato (Solanum lycopersicum) and in a related wild species (S. habrochaites) is provided. RESULTS: In both species, the development of these structures follows a highly reproducible cell division pattern. The two species differ in the shape of the trichome head which is round in S. habrochaites and like a four-leaf clover in S. lycopersicum, correlating with the presence of a large intercellular cavity in S. habrochaites where the produced metabolites accumulate. In both species, the junction between the intermediate cell and the four glandular cells constitute a breaking point facilitating the decapitation of the trichome and thereby the quick release of the metabolites. A strongly auto-fluorescent compound transiently accumulates in the early stages of development suggesting a potential role in the differentiation process. Finally, immuno-labelling with antibodies recognizing specific cell wall components indicate a key role of pectin and arabinogalactan components in the differentiation of type VI trichomes. CONCLUSIONS: Our observations explain the adaptive morphologies of type VI trichomes for metabolite storage and release and provide a framework for further studies of these important metabolic cellular factories. This is required to better exploit their potential, in particular for the breeding of pest resistance in tomato.

A starch-based implant as a controlled drug release system: Non-invasive in vivo characterization using multispectral fluorescence imaging.

Solid implants are parenteral depot systems that can provide a controlled release of drugs in the desired body area over a few days to months. Finding an alternative for the two most commonly used polymers in the production of parenteral depot systems, namely Poly-(lactic acid) (PLA) and Poly-(lactide-co-glycolide) (PLGA), is of great importance due to their certain drawbacks. Our previous study showed the general suitability of starch-based implants for controlled drug release system. In this study, the system is further characterized and the release kinetics are investigated in vitro and in vivo by fluorescence imaging (FI). ICG and DiR, two fluorescent dyes with different hydrophobicity serving as a model for hydrophilic and hydrophobic drugs, have been used. In addition to 2D FI, 3D reconstructions of the starch implant were also used to assess the release kinetics in 3D mode. The in vitro and in vivo studies showed a fast release of ICG and a sustained release of DiR over 30 days from the starch-based implant. No treatment-related adverse effects were observed in mice. Our results indicate the promising potential of the biodegradable biocompatible starch-based implant for the controlled release of hydrophobic drugs.

Tissue response to biphasic calcium phosphate covalently modified with either heparin or hyaluronic acid in a mouse subcutaneous implantation model.

Biphasic calcium phosphate (BCP) materials are widely employed as bone substitute materials due to their resorption/degradation properties. Inflammation after implantation of such materials represents a prerequisite for bone tissue repair and regeneration but can be also problematic if it is not only transient and if it is followed by fibrosis and scarring. Here, we modified BCP covalently with hyaluronan (HA) and heparin (Hep), glycosaminoglycans that possess anti-inflammatory properties. Beside the characterization of particle surface properties, the focus was on in vivo tissue response after subcutaneous implantation in mice. Histological analysis revealed a decrease in signs of inflammatory response to BCP when modified with either HA or Hep. Reduced vascularization after 30 days was noticed when BCP was modified with either HA or Hep with greater cellularity in all examined time points. Compared to plain BCP, expression of endothelial-related genes Flt1 and Vcam1 was higher in BCP-HA and BCP-Hep group at day 30. Expression of osteogenesis-related genes Sp7 and Bglap after 30 days was the highest in BCP group, followed by BCP-Hep, while the lowest expression was in BCP-HA group which correlates with collagen amount. Hence, coating of BCP particles with HA seems to suppress inflammatory response together with formation of new bone-like tissue, while the presence of Hep delays the onset of inflammatory response but permits osteogenesis in this subcutaneous bone-forming model. Transferring the results of this study to other coated materials intended for biomedical application may also pave the way to reduction of inflammation after their implantation.

Extraction and characterization of celluloses from various plant byproducts.

Celluloses were extracted from teff straw (TS), enset fiber (EF), sugarcane bagasse (SB) and coffee hull (CH) agro-industrial byproducts generated in large quantities in Ethiopia. The present study aimed to explore these plant byproducts as alternative sources of cellulose for potential industrial applications, using various eco-friendly chlorine-free treatment conditions to obtain an optimum cellulose extraction condition. The byproducts and the as-extracted celluloses were analyzed for chemical compositions, yield, chemical functionality, crystallinity, thermal stability and morphology. EF yielded the highest cellulose content (60.0%), whereas CH the least (35.5%). FTIR spectra and ESEM morphological studies of the celluloses indicated progressive removal of non-cellulosic constituents. XRD analyses showed EF cellulose had the highest crystallinity index (CrI) (85.56%), crystallite size (5.52 nm), and proportion of crystallite interior chains of 200 plane (0.629), exhibiting unique physicochemical properties. The byproducts and the as-extracted celluloses showed Cellulose Iß crystal lattice, while celluloses from EF and SB also displayed (partial) polymorphic transition into Cellulose II. TGA studies revealed enhanced stability of the as-extracted celluloses. On the basis of the physicochemical characteristics of the celluloses, all the byproducts studied could be considered as alternative sources of cellulose for potential value-added industrial applications.

In situ Gelling Amphotericin B Nanofibers: A New Option for the Treatment of Keratomycosis.

The purpose of our research was the development of Amphotericin B-loaded in situ gelling nanofibers for the treatment of keratomycosis. Different formulation strategies were applied to increase the drug load of the sparingly water-soluble Amphotericin B in electrospun Gellan Gum/Pullulan fibers. These include bile salt addition, encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and formation of a polymeric Amphotericin B polyelectrolyte complex. The Amphotericin B polyelectrolyte complex (AmpB-Eu L) performed best and was very effective against the fungal strain Issatchenkia orientalis in vitro. The complex was characterized in detail by attenuated total reflection infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. A heat induced stress test was carried out to ensure the stability of the polyelectrolyte complex. To gain information about the cellular tolerance of the developed polyelectrolyte complex a new, innovative multilayered-stratified human cornea cell model was used for determination of the cellular toxicity in vitro. For a safe therapy, the applied ophthalmic drug delivery system has to be sterile. Sterilization by electron irradiation caused not degradation of pure Amphotericin B and also for the bile salt complex. Furthermore, the developed Amphotericin B polyelectrolyte complex was not degraded by the irradiation process. In conclusion, a new polyelectrolyte Amphotericin B complex has been found which retains the antifungal activity of the drug with sufficient stability against irradiation-sterilization induced drug degradation. Furthermore, in comparison with the conventional used eye drop formulation, the new AmpB-complex loaded nanofibers were less toxic to cornea cells in vitro. Electrospinning of the Amphotericin B polyelectrolyte complex with Gellan Gum/ Pullulan leads to the formation of nanofibers with in situ gelling properties, which is a new and promising option for the treatment of keratomycosis.

Electrospun Nimodipine-loaded fibers for nerve regeneration: Development and in vitro performance.

Nimodipine is a 1,4-Dihydropyridine type calcium antagonist routinely used to control blood pressure and reduce the risk of secondary ischemia after aneurismal subarachnoid hemorrhage. Additionally, Nimodipine has unique neuroprotective properties. With respect to brain related applications, the full potential of the desired local effect can often not be realized after systemic administration due to systemic side effects. Therefore, it was our aim to develop a biodegradable drug delivery system for the local controlled release of the drug inside the brain. As a suitable and biodegradable system we successfully electrospun PLGA fibers containing 1 and 10% drug. The results of DSC and X-Ray diffractometry measurements indicate that Nimodipine was incorporated in the polymer matrix in the amorphous state. No drug recrystallization was detected for up to 6 months. Electron-beam sterilization was tried but reduced the drug content of the fiber mats considerably. A sustained drug release over 4-8 days was observed, highly depended on release conditions. The Nimodipine fiber mats exhibited no cell toxicity. In contrast, the electrospun fibers were able to significantly reduce cell death in in vitro cell models of oxidative, osmotic and heat-induced cell stress in Schwann cells, neuronal cells as well as immortalized and primary astrocytes. Therefore, electrospun Nimodipine loaded PLGA fibers represent a promising drug delivery system to realize the druǵs benefits for its intracranial use.

Electrospun nanofibers - A promising solid in-situ gelling alternative for ocular drug delivery.

A serious problem of the treatment of eye diseases is the very short residence time of the drug. The majority of the drug is cleared within few seconds due to the poor capability of the eye to accommodate additional liquids. We developed a new ocular drug delivery system, which is applied in dry form and forms immediately a gel after administration. The system is based on gellan gum/pullulan electrospun nanofibers. The rheological behavior of the spinning solution was investigated followed by further characterization of the in situ formed gel. Three-dimensional X-ray imaging with nanometric resolution (nano-CT) and electron scanning microscopy were used for a detailed characterization of the diameter and alignment of the fibers. A high porosity (87.5 ± 0.5%) and pore interconnectivity (99%) was found. To ensure a good fit to the eye anatomy, the prepared fibers were shaped into curved geometries. Additionally, a new innovative moistening chamber for the in vitro determination of the ocular residence time in porcine eyes was developed which mimics the tear turnover. A clear prolongation of the fluorescein residence time compared to conventional eye drops was achieved with the application of the curved nanofiber in situ gelling mat. In summary, the developed in situ gelling system with adapted geometry is a promising alternative system for ocular drug delivery.

Contact-Triggered Lipofection from Multilayer Films Designed as Surfaces for in Situ Transfection Strategies in Tissue Engineering.

Biomaterials, which release active compounds after implantation, are an essential tool for targeted regenerative medicine. In this study, thin multilayer films loaded with lipid/DNA complexes (lipoplexes) were designed as surface coatings for in situ transfection applicable in tissue engineering and regenerative medicine. The film production and embedding of lipoplexes were based on the layer-by-layer (LbL) deposition technique. Hyaluronic acid (HA) and chitosan (CHI) were used as the polyelectrolyte components. The embedded plasmid DNA was complexed using a new designed cationic lipid formulation, namely, OH4/DOPE 1/1, the advantageous characteristics of which have been proven already. Three different methods were tested regarding its efficiency of lipid and DNA deposition. Therefore, several surface specific analytics were used to characterize the LbL formation, the lipid DNA embedding, and the surface characteristics of the multilayer films, such as fluorescence microscopy, surface plasmon resonance spectroscopy, ellipsometry, zeta potential measurements, atomic force microscopy, and scanning electron microscopy. Interaction studies were conducted for optimized lipoplex-loaded polyelectrolyte multilayers (PEMs) that showed an efficient attachment of C2C12 cells on the surface. Furthermore, no acute toxic effects were found in cell culture studies, demonstrating biocompatibility. Cell culture experiments with C2C12 cells, a cell line which is hard to transfect, demonstrated efficient transfection of the reporter gene encoding for green fluorescent protein. In vivo experiments using the chicken embryo chorion allantois membrane animal replacement model showed efficient gene-transferring rates in living complex tissues, although the DNA-loaded films were stored over 6 days under wet and dried conditions. Based on these findings, it can be concluded that OH4/DOPE 1/1 lipoplex-loaded PEMs composed of HA and CHI can be an efficient tool for in situ transfection in regenerative medicine.

Effect of Different Crosslinking Strategies on Physical Properties and Biocompatibility of Freestanding Multilayer Films Made of Alginate and Chitosan.

Freestanding multilayer films prepared by layer-by-layer technique have attracted interest as promising materials for wound dressings. The goal is to fabricate freestanding films using chitosan (CHI) and alginate (ALG) including subsequent crosslinking to improve the mechanical properties of films while maintaining their biocompatibility. Three crosslinking strategies are investigated, namely use of calcium ions for crosslinking ALG, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide for crosslinking ALG with CHI, and Genipin for crosslinking chitosan inside the films. Different characteristics, such as surface morphology, wettability, swelling, roughness, and mechanical properties are investigated showing that films became thinner, exhibited rougher surfaces, had lower water uptake, and increased mechanical strength after crosslinking. Changes of wettability are moderate and dependent on the crosslinking method. In vitro cytotoxicity and cell attachment studies with human dermal fibroblasts show that freestanding CHI-ALG films represent a poorly adhesive substratum for fibroblasts, while studies using incubation of plastic-adherent fibroblast beneath floating films show no signs of cytotoxicity in a time frame of 7 days. Results from cell experiments combined with film characteristics after crosslinking, indicate that crosslinked freestanding films made of ALG and CHI may be interesting candidates for wound dressings.

Micromorphometrical analysis of conventional osteotomy techniques and ultrasonic osteotomy at the rabbit skull.

OBJECTIVES: The ultrasonic osteotome, which was recently introduced, is an alternative to conventional methods of osteotomy. The aim of the present study was to establish the differences between three osteotomy techniques and to perform a quantitative roughness analysis of the osteotomized bone surfaces. MATERIALS AND METHODS: Fresh bony samples of standardized size were taken from the rabbit skull. The techniques used were as follows: reciprocate micro-saw, Lindemann bur, ultrasonic osteotome with the two insert tips OT6 (rough) and OT7 (fine). The prepared surfaces were examined by light microscopy, environmental surface electron microscopy (ESEM) and by confocal laser scanning microscopy (CLSM). RESULTS: It was difficult to distinguish between cortical and cancellous bone after using the conventional osteotomy technique. The ultrasonic technique preserved the original structure of the bone. The values observed for superficial roughness were as follows: 3.97 microm (micro-saw), 5.7 microm (Lindemann bur), 2.48 microm (OT7) and 3 microm (OT6). There were statistical differences between the values of the bur and insert tip OT6 (P=0.015) as well as between the bur and insert tip OT7 (P=0.003). CONCLUSIONS: In the present study micromorphological differences after using various osteotomy techniques could be clearly identified.

3D Powder Printed Bioglass and ß-Tricalcium Phosphate Bone Scaffolds.

The use of both bioglass (BG) and ß tricalcium phosphate (ß-TCP) for bone replacement applications has been studied extensively due to the materials' high biocompatibility and ability to resorb when implanted in the body. 3D printing has been explored as a fast and versatile technique for the fabrication of porous bone scaffolds. This project investigates the effects of using different combinations of a composite BG and ß-TCP powder for 3D printing of porous bone scaffolds. Porous 3D powder printed bone scaffolds of BG, ß-TCP, 50/50 BG/ß-TCP and 70/30 BG/ß-TCP compositions were subject to a variety of characterization and biocompatibility tests. The porosity characteristics, surface roughness, mechanical strength, viability for cell proliferation, material cytotoxicity and in vitro bioactivity were assessed. The results show that the scaffolds can support osteoblast-like MG-63 cells growth both on the surface of and within the scaffold material and do not show alarming cytotoxicity; the porosity and surface characteristics of the scaffolds are appropriate. Of the two tested composite materials, the 70/30 BG/ß-TCP scaffold proved to be superior in terms of biocompatibility and mechanical strength. The mechanical strength of the scaffolds makes them unsuitable for load bearing applications. However, they can be useful for other applications such as bone fillers.

Cytometric patterns reveal growth states of Shewanella putrefaciens.

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.

Toward a detailed characterization of oil adsorbates as "solid liquids".

Solid lipid formulation systems are used to overcome oral bioavailability problems of poorly water-soluble drugs. One promising process is the conversion of a liquid lipid system in a free flowing powder by use of adsorbing excipients. The aim of this study was the detailed characterization of solid-liquid interactions in oil adsorbed to Fujicalin and Neusilin which were manufactured by means of dual asymmetric centrifugation or conventional mortar/pestle blending. The adsorption strength of the excipients was investigated by Benchtop-NMR and ESR spectroscopy revealing the highest adsorption power for the Neusilin products. The adsorbate production methods as well as the storage of the excipients impact their adsorption properties. Environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM) show that dual asymmetric centrifugation leads to a smoothing of the particle surface, whereas the mortar/pestle blending results in an uneven surface and particle destruction. The oil distribution at the particles is inhomogeneous for both production methods. The micropolarity of the adsorbed oil was investigated by ESR spectroscopy and multispectral fluorescence imaging. The adsorbing process on Neusilin leads to an increased micropolarity of the oil component. The release of the oil component in aqueous media could be verified by Benchtop-NMR and multispectral fluorescence imaging.

Monitoring of dissolution induced changes in film coat composition by 1H NMR spectroscopy and SEM.

In our study we investigated the release mechanisms of coated oral dosage forms more deeply. The aim of the study was to monitor the buffer induced leaching out of water soluble compounds from Kollicoat SR films and to relate this process to the film properties and the release kinetics. Propranolol HCl tablets were coated with different amounts and ratios of polyvinyl acetate (Kollicoat SR) and poly(vinyl alcohol)-poly(ethylene glycol)-graft-copolymer (Kollicoat IR). Polyvinyl pyrrolidone (Kollidon 30) was added as a second water soluble polymer and triacetin as a plasticizer. In addition to kinetics of the drug release, the films were studied by SEM and 1H NMR spectroscopy. SEM micrographs revealed morphological changes of the tablet surface that were related to an alteration in film coat composition. The described 1H NMR method provided the opportunity to quantify the leaching of Kollicoat IR, Kollidon 30 and triacetin. Drug release kinetics were related to dissolution induced changes in coating composition. Permeability of the film coat increased, when about 90% of the water soluble polymers and plasticizer triacetin were leached out of the film coat.

Modeling of optical properties of silver-doped nanocomposite glasses modified by electric-field-assisted dissolution of nanoparticles.

It has been demonstrated recently that silver nanoparticles embedded in a glass matrix can be dissolved by the combination of an intense dc electric field and moderately elevated temperature. In an intermediate state of this process percolated silver layers inside the glass can also occur. These structural modifications significantly modify the optical behavior of the glass, suggesting an interesting perspective for the engineering of optical properties of this kind of metallodielectric materials. We present the optical characterization of silver-doped glasses subjected to the electric-field-assisted dissolution of nanoparticles. The characterization is performed by means of fitting spectrophotometric measurements. The optical properties of the investigated samples are described in terms of the interference between the light reflected from the glass surface and the light reflected from a buried silver-containing layer formed in the depths of the glass. The analysis of the data reveals a porosity of the glass in the region where the nanoparticles are dissolved that can be attributed to the presence of residual nanopores.