Controlled formation of chitosan particles by a clock reaction.

Clock reactions allow precise control of chemical composition in the time domain. Such nonlinear chemical systems have recently been introduced to mimic the self-assembly pathways common in living organisms. Here, we demonstrate the use of a clock reaction to trigger the formation of polymeric nanoparticles. By adjusting the delay of a formaldehyde clock reaction, we controlled the precipitation of chitosan to form particles with sizes tunable in a wide range (from about 200 to 600 nm diameter). The chemical structure of chitosan was not significantly perturbed by the clock reagents.

Harvesting pre-polarized macrophages using thermo-responsive substrates.

In the cell culture environment macrophages are highly adherent cells. Currently used methods to harvest macrophages have the disadvantage of reducing cell viability and their ability to re-attach after seeding. Although thermo-responsive surfaces have been employed to harvest cell sheets no reports are available to use these to harvest (pre-polarized) macrophages. We show that this method significantly improves the yield of living macrophages and percentage of subsequent cell reattachment, whilst having a minimal effect on the cell phenotype.

The agar diffusion scratch assay--A novel method to assess the bioactive and cytotoxic potential of new materials and compounds.

A profound in vitro evaluation not only of the cytotoxic but also of bioactive potential of a given compound or material is crucial for predicting potential effects in the in vivo situation. However, most of the current methods have weaknesses in either the quantitative or qualitative assessment of cytotoxicity and/or bioactivity of the test compound. Here we describe a novel assay combining the ISO 10993-5 agar diffusion test and the scratch also termed wound healing assay. In contrast to these original tests this assay is able to detect and distinguish between cytotoxic, cell migration modifying and cytotoxic plus cell migration modifying compounds, and this at higher sensitivity and in a quantitative way.

Effect of particle agglomeration in nanotoxicology.

The emission of engineered nanoparticles (ENPs) into the environment in increasing quantity and variety raises a general concern regarding potential effects on human health. Compared with soluble substances, ENPs exhibit additional dimensions of complexity, that is, they exist not only in various sizes, shapes and chemical compositions but also in different degrees of agglomeration. The effect of the latter is the topic of this review in which we explore and discuss the role of agglomeration on toxicity, including the fate of nanomaterials after their release and the biological effects they may induce. In-depth investigations of the effect of ENP agglomeration on human health are still rare, but it may be stated that outside the body ENP agglomeration greatly reduces human exposure. After uptake, agglomeration of ENPs reduces translocation across primary barriers such as lungs, skin or the gastrointestinal tract, preventing exposure of "secondary" organs. In analogy, also cellular ENP uptake and intracellular distribution are affected by agglomeration. However, agglomeration may represent a risk factor if it occurs after translocation across the primary barriers, and ENPs are able to accumulate within the tissue and thus reduce clearance efficiency.

In vitro investigations of a novel wound dressing concept based on biodegradable polyurethane.

Non-healing and partially healing wounds are an important problem not only for the patient but also for the public health care system. Current treatment solutions are far from optimal regarding the chosen material properties as well as price and source. Biodegradable polyurethane (PUR) scaffolds have shown great promise for in vivo tissue engineering approaches, but accomplishment of the goal of scaffold degradation and new tissue formation developing in parallel has not been observed so far in skin wound repair. In this study, the mechanical properties and degradation behavior as well as the biocompatibility of a low-cost synthetic, pathogen-free, biocompatible and biodegradable extracellular matrix mimicking a PUR scaffold was evaluated in vitro. The novel PUR scaffolds were found to meet all the requirements for optimal scaffolds and wound dressings. These three-dimensional scaffolds are soft, highly porous, and form-stable and can be easily cut into any shape desired. All the material formulations investigated were found to be nontoxic. One formulation was able to be defined that supported both good fibroblast cell attachment and cell proliferation to colonize the scaffold. Tunable biodegradation velocity of the materials could be observed, and the results additionally indicated that calcium plays a crucial role in PUR degradation. Our results suggest that the PUR materials evaluated in this study are promising candidates for next-generation wound treatment systems and support the concept of using foam scaffolds for improved in vivo tissue engineering and regeneration.

Improving cell adhesion: development of a biosensor for cell behaviour monitoring by surface grafting of sulfonic groups onto a thermoplastic polyurethane.

The surface properties of a material in combination with the mechanical properties are responsible for the material performance in a biological environment as well as the behaviour of the cells which contact with the material. Surface properties such as chemical, physical, biological play an important role in the biomaterials filed. In this work, the surface of a thermoplastic polyurethane film (Elastollan(®)1180A50) was tailored with sulfonic groups by grafting [2-(methacryloxyl)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (SB) after a previous surface activation either by Argon plasma or by ultra-violet irradiation. This surface modification had the purpose of improving cell adhesion in order to develop a biosensor able to monitor cell behaviour. The surfaces were characterized by X-ray photoelectron spectroscopy, by atomic force microscopy and by contact angle measurements in order to evaluate the efficiency of the modification. Additionally, blood compatibility studies and cell adhesion tests with human bone marrow cells were performed. These methods allowed the grafting of SB and the results indicate that a higher density of grafting was achieved with previous surface plasma treatment than with UV irradiation. However, for both techniques, the presence of SB functional groups led to a decrease of hydrophobicity and roughness of the surface, together with an improvement of the materials biological performance.

From implantation to degradation - are poly (l-lactide)/multiwall carbon nanotube composite materials really cytocompatible?

Poly (l-lactide)'s (PLLA) biodegradable properties are of special value in orthopaedic applications, but its mechanical strength limits its usage. To overcome this PLLA can be reinforced by multiwall carbon nanotubes (MWCNT). In this study the PLLA and MWCNT were combined to prepare nanostructured composites (nanocomposite) at 0, 0.1, 0.5 and 1wt.% reinforcement. The in vitro biocompatibility of these PLLA/MWCNT nanocomposites was evaluated taking into account the various stages of implantation including nanocomposite degradation. PLLA/MWCNT nanocomposites were highly biocompatible with human bone marrow stromal cells (HBMC). The potential surface degradation product, MWCNT, did not induce toxic responses on HBMC. However, the combination of MWCNT with lactic acid, resembling release after bulk degradation, significantly inhibited HBMC proliferation and activity. This study demonstrates the importance of comprehensive evaluations of novel materials for medical applications in predicting possible adverse effects during nanocomposite degradation. FROM THE CLINICAL EDITOR: This study scrutinizes the cytocompatibility of poly-L-lactide reinforced by multiwall carbon nanotubes, and concludes that the combination of MWCNT with lactic acid significantly inhibited human bone marrow stromal cell proliferation and activity, highlighting the importance of comprehensive evaluations of novel materials.

Addition of nanoscaled bioinspired surface features: A revolution for bone related implants and scaffolds?

Our expanding ability to handle the "literally invisible" building blocks of our world has started to provoke a seismic shift on the technology, environment and health sectors of our society. During the last two decades, it has become increasingly evident that the "nano-sized" subunits composing many materials­living, natural and synthetic­are becoming more and more accessible for predefined manipulations at the nanosize scale. The use of equally nanoscale sized or functionalised tools may, therefore, grant us unprecedented prospects to achieve many therapeutic aims. In the past decade it became clear that nano-scale surface topography significantly influences cell behaviour and may, potentially, be utilised as a powerful tool to enhance the bioactivity and/ or integration of implanted devices. In this review, we briefly outline the state of the art and some of the current approaches and concepts for the future utilisation of nanotechnology to create biomimetic implantable medical devices and scaffolds for in vivo and in vitro tissue engineering,with a focus on bone. Based on current knowledge it must be concluded that not the materials and surfaces themselves but the systematic biological evaluation of these new material concepts represent the bottleneck for new biomedical product development based on nanotechnological principles.

Evaluation of early stage human bone marrow stromal proliferation, cell migration and osteogenic differentiation on µ-MIM structured stainless steel surfaces.

It is well established that surface topography greatly affect cell-surface interactions. In a recent study we showed that microstructured stainless steel surfaces characterized by the presence of defined hexagonally arranged hemisphere-like structures significantly affected cell architecture (shape and focal adhesion size) of primary human bone mesenchymal stromal cells. This study aimed at further investigating the influence these microstructures (microcline protruding hemispheres) on critical aspects of cell behaviour namely; proliferation, migration and osteogenic differentiation. As with previously reported data, we used primary human bone mesenchymal stromal cells to investigate such effects at an early stage in vitro. Cells of different patients were utilised for cell migration studies. Our data showed that an increase in cell proliferation was exhibited as a function of surface topography (hemispheres). Cell migration velocity also varied as a function of surface topography on patient specific basis and seems to relate to the differentiated state of the seeded cell population (as demonstrated by bALP positivity). Osteogenic differentiation, however, did not exhibit significant variations (both up and down-regulation) as a function of both surface topography and time in culture.

Human triple cell co-culture for evaluation of bone implant materials.

Central to the formation of tissue at implant surfaces are the interactions between multiple cell types including fibroblasts, endothelial cells and, in the case of bone, cells of the osteoblastic lineage. To date the importance of population dynamics and interactions have been largely neglected in the in vitro evaluation of biomaterials. To fill this gap we have developed a co-culture system using 3 cell types, primary human bone marrow stromal cells (HBMC), microvascular endothelial cells (HMVEC) and abdominal dermal fibroblasts (HDF). Proliferation of each cell type separately and differentiation of HBMC were determined by flow cytometry analysis. The medium used promoted HBMC differentiation toward osteoblasts without affecting the state of differentiation of HDF and HMVEC. Furthermore, HBMC are strongly affected by HDF and HMVEC, and vice versa. When used on a titanium coated substrate the triple cell culture system identified preferential HBMC proliferation relative to HDF if HMVEC was present. This developed culture system represents a new, optimised and potentially predictive approach to evaluate biomaterial biocompatibility early in development.

Surface microstructures on planar substrates and textile fibers guide neurite outgrowth: a scaffold solution to push limits of critical nerve defect regeneration?

The treatment of critical size peripheral nerve defects represents one of the most serious problems in neurosurgery. If the gap size exceeds a certain limit, healing can't be achieved. Connection mismatching may further reduce the clinical success. The present study investigates how far specific surface structures support neurite outgrowth and by that may represent one possibility to push distance limits that can be bridged. For this purpose, growth cone displacement of fluorescent embryonic chicken spinal cord neurons was monitored using time-lapse video. In a first series of experiments, parallel patterns of polyimide ridges of different geometry were created on planar silicon oxide surfaces. These channel-like structures were evaluated with and without amorphous hydrogenated carbon (a-C:H) coating. In a next step, structured and unstructured textile fibers were investigated. All planar surface materials (polyimide, silicon oxide and a-C:H) proved to be biocompatible, i.e. had no adverse effect on nerve cultures and supported neurite outgrowth. Mean growth cone migration velocity measured on 5 minute base was marginally affected by surface structuring. However, surface structure variability, i.e. ridge height, width and inter-ridge spacing, significantly enhanced the resulting net velocity by guiding the growth cone movement. Ridge height and inter-ridge distance affected the frequency of neurites crossing over ridges. Of the evaluated dimensions ridge height, width, and inter-ridge distance of respectively 3, 10, and 10 µm maximally supported net axon growth. Comparable artificial grooves, fabricated onto the surface of PET fibers by using an excimer laser, showed similar positive effects. Our data may help to further optimize surface characteristics of artificial nerve conduits and bioelectronic interfaces.

In vitro bioactivity of micro metal injection moulded stainless steel with defined surface features.

Micrometre- and nanometre-scale surface structuring with ordered topography features may dramatically enhance orthopaedic implant integration. In this study we utilised a previously optimised micron metal injection moulding (µ-MIM) process to produce medical grade stainless steel surfaces bearing micrometre scale, protruding, hemispheres of controlled dimensions and spatial distribution. Additionally, the structured surfaces were characterised by the presence of submicrometre surface roughness resulting from metal grain boundary formation. Following cytocompatibility (cytotoxicity) evaluation using 3T3 mouse fibroblast cell line, the effect on primary human cell functionality was assessed focusing on cell attachment, shape and cytoskeleton conformation. In this respect, and by day 7 in culture, significant increase in focal adhesion size was associated with the microstructured surfaces compared to the planar control. The morphological conformation of the seeded cells, as revealed by fluorescence cytoskeleton labelling, also appeared to be guided in the vertical dimension between the hemisphere bodies. Quantitative evaluation of this guidance took place using live cytoplasm fluorescence labelling and image morphometry analysis utilising both, compactness and elongation shape descriptors. Significant increase in cell compactness was associated with the hemisphere arrays indicating collective increase in focused cell attachment to the hemisphere bodies across the entire cell population. Micrometre-scale hemisphere array patterns have therefore influenced cell attachment and conformation. Such influence may potentially aid in enhancing key cellular events such as, for example, neo-osteogenesis on implanted orthopaedic surfaces.

Evaluation of biocompatibility using in vitro methods: interpretation and limitations.

The in vitro biocompatibility of novel materials has to be proven before a material can be used as component of a medical device. This must be done in cell culture tests according to internationally recognized standard protocols. Subsequently, preclinical and clinical tests must be performed to verify the safety of the new material and device. The present chapter focuses on the first step, the in vitro testing according to ISO 10993-5, and critically discusses its limited significance. Alternative strategies and a brief overview of activities to improve the current in vitro tests are presented in the concluding section.

Surface grafting of a thermoplastic polyurethane with methacrylic acid by previous plasma surface activation and by ultraviolet irradiation to reduce cell adhesion.

The material performance, in a biological environment, is mainly mediated by its surface properties and by the combination of chemical, physical, biological, and mechanical properties required, for a specific application. In this study, the surface of a thermoplastic polyurethane (TPU) material (Elastollan(®)1180A50) was activated either by plasma or by ultra-violet (UV) irradiation. After surface activation, methacrylic acid (MAA) was linked to the surface of TPU in order to improve its reactivity and to reduce cell adhesion. Grafted surfaces were evaluated by X-ray photoelectron spectroscopy (XPS), by atomic force microscopy (AFM) and by contact angle measurements. Blood compatibility studies and cell adhesion tests with human bone marrow cells (HBMC) were also performed. If was found that UV grafting method led to better results than the plasma activation method, since cell adhesion was reduced when methacrylic acid was grafted to the TPU surface by UV.

Novel in vitro co-culture methodology to investigate heterotypic cell-cell interactions.

Cell-cell interactions are of crucial importance for the formation of tissues, homeostasis and regeneration processes as well as reactions on foreign bodies including implants. So far, however, the importance of heterotypic cell-cell interactions in the in vitro evaluation of implant surfaces has been largely neglected. This work aims to develop an in vitro methodology that enables the in-depth investigation of heterotypic cell-cell interactions in a mixed co-culture system, and to validate it with a primary adult human bone-derived osteoblast cells (HBCs) - abdominal fibroblasts (HAFs) system. The methodology proposed combines a simple live labelling step, semiautomated fluorescence image acquisition and analysis to characterize the interactions between different cell types (cell population dynamics) in co-culture in terms of cell proliferation and cell spatial distribution of each cell type. In this co-culture system, direct cell-cell contacts between the two cell types were permitted while the determination of cell-type specific responses could still be elucidated. We could show that HAF proliferation was reduced in a way negatively correlated with the seeding HBC/HAF ratio, i.e., a high proportion of HBC in the co-culture had an inhibitory effect on HAF proliferation. In all cultures segregation was found after 4 and 7 days of co-culture. HBCs were segregated at low ratios while HAFs were segregated at high ratios. Cell-cell distances depended on the total cell number in the co-culture but the dependence was different for each cell type.

Single walled carbon nanotubes (SWCNT) affect cell physiology and cell architecture.

Single walled carbon nanotubes (SWCNT) find their way in various industrial applications. Due to the expected increased production of various carbon nanotubes and nanoparticle containing products, exposure to engineered nanoparticles will also increase dramatically in parallel. In this study the effects of SWCNT raw material and purified SWCNT (SWCNT bundles) on cell behaviour of mesothelioma cells (MSTO-211H) and on epithelial cells (A549) had been investigated. The effect on cell behaviour (cell proliferation, cell activity, cytoskeleton organization, apoptosis and cell adhesion) were dependent on cell type, SWCNT quality (purified or not) and SWCNT concentration.

Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress.

The chemical and catalytic activity of nanoparticles has strongly contributed to the current tremendous interest in engineered nanomaterials and often serves as a guiding principle for the design of functional materials. Since it has most recently become evident that such active materials can enter into cells or organisms, the present study investigates the level of intracellular oxidations after exposure to iron-, cobalt-, manganese-, and titania-containing silica nanoparticles and the corresponding pure oxides in vitro. The resulting oxidative stress was quantitatively measured as the release of reactive oxygen species (ROS). The use of thoroughly characterized nanoparticles of the same morphology, comparable size, shape, and degree of agglomeration allowed separation of physical (rate of particle uptake, agglomeration, sedimentation) and chemical effects (oxidations). Three sets of control experiments elucidated the role of nanoparticles as carriers for heavy metal uptake and excluded a potential interference of the biological assay with the nanomaterial. The present results indicate that the particles could efficiently enter the cells by a Trojan-horse type mechanism which provoked an up to eight times higher oxidative stress in the case of cobalt or manganese if compared to reference cultures exposed to aqueous solutions of the same metals. A systematic investigation on iron-containing nanoparticles as used in industrial fine chemical synthesis demonstrated that the presence of catalytic activity could strongly alter the damaging action of a nanomaterial. This indicates that a proactive development of nanomaterials and their risk assessment should consider chemical and catalytic properties of nanomaterials beyond a mere focus on physical properties such as size, shape, and degree of agglomeration.

The degree and kind of agglomeration affect carbon nanotube cytotoxicity.

The urgent need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a basis for comparison to existing epidemiological data, we have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human MSTO-211H cells. Non-cytotoxic polyoxyethylene sorbitan monooleate was found to well-disperse CNT. In the present study, the cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference. While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibres at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.

Identification of neurotoxic chemicals in cell cultures.

In order to identify the neurotoxic potential of drugs or chemicals in vitro, a combination of different in vitro cell culture tests is required. Depending on the type and use of a given test compound, a sequential exposure of freshly isolated and cultured hepatocytes and chicken brain cells is suitable. In order to find out more about the stability of liver-derived metabolites, co-cultures are appropriate. In order to determine how metabolites enter the brain, a combination of an in vitro system which mimics the blood-brain barrier and choroid plexus is proposed.

In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility.

Early indicators for nanoparticle-derived adverse health effects should provide a relative measure for cytotoxicity of nanomaterials in comparison to existing toxicological data. We have therefore evaluated a human mesothelioma and a rodent fibroblast cell line for in vitro cytotoxicity tests using seven industrially important nanoparticles. Their response in terms of metabolic activity and cell proliferation of cultures exposed to 0-30 ppm nanoparticles (microg g(-1)) was compared to the effects of nontoxic amorphous silica and toxic crocidolite asbestos. Solubility was found to strongly influence the cytotoxic response. The results further revealed a nanoparticle-specific cytotoxic mechanism for uncoated iron oxide and partial detoxification or recovery after treatment with zirconia, ceria, or titania. While in vitro experiments may never replace in vivo studies, the relatively simple cytotoxic tests provide a readily available pre-screening method.