Laser-induced topographies enable the spatial patterning of co-cultured peripheral nervous system cells.

The peripheral nervous system comprises glia and neurons that receive the necessary cues for their adhesion and proliferation from their extracellular milieu. In this study, a spatial platform of pseudoperiodic morphologies including patterns of nano- and micro- structures on Si were developed via direct ultrafast-laser structuring and were used as substrates for the patterning of co-cultured neuronal cells. The response of murine Schwann (SW10) and Neuro2a (N2a) cells were investigated both in monocultures and in a glia and neuronal co-culture system. Our results denoted that different types of neural tissue cells respond differently to the underlying topography, but furthermore, the presence of the glial cells alters the adhesion behavior of the neuronal cells in their co-culture. Therefore, we envisage that direct laser structuring that enables spatial patterning of the cells of the nervous system in a controllable manner according to the research needs, could in the future be a useful tool for understanding neural network interfaces and their electrical activity, synaptic processes and myelin formation.

Biofabrication for neural tissue engineering applications.

Unlike other tissue types, the nervous tissue extends to a wide and complex environment that provides a plurality of different biochemical and topological stimuli, which in turn defines the advanced functions of that tissue. As a consequence of such complexity, the traditional transplantation therapeutic methods are quite ineffective; therefore, the restoration of peripheral and central nervous system injuries has been a continuous scientific challenge. Tissue engineering and regenerative medicine in the nervous system have provided new alternative medical approaches. These methods use external biomaterial supports, known as scaffolds, to create platforms for the cells to migrate to the injury site and repair the tissue. The challenge in neural tissue engineering (NTE) remains the fabrication of scaffolds with precisely controlled, tunable topography, biochemical cues, and surface energy, capable of directing and controlling the function of neuronal cells toward the recovery from neurological disorders and injuries. At the same time, it has been shown that NTE provides the potential to model neurological diseases in vitro, mainly via lab-on-a-chip systems, especially in cases for which it is difficult to obtain suitable animal models. As a consequence of the intense research activity in the field, a variety of synthetic approaches and 3D fabrication methods have been developed for the fabrication of NTE scaffolds, including soft lithography and self-assembly, as well as subtractive (top-down) and additive (bottom-up) manufacturing. This article aims at reviewing the existing research effort in the rapidly growing field related to the development of biomaterial scaffolds and lab-on-a-chip systems for NTE applications. Besides presenting recent advances achieved by NTE strategies, this work also delineates existing limitations and highlights emerging possibilities and future prospects in this field.

Cell patterning via laser micro/nano structured silicon surfaces.

The surface topography of biomaterials can have an important impact on cellular adhesion, growth and proliferation. Apart from the overall roughness, the detailed morphological features, at all length scales, significantly affect the cell-biomaterial interactions in a plethora of applications including structural implants, tissue engineering scaffolds and biosensors. In this study, we present a simple, one-step direct laser patterning technique to fabricate nanoripples and dual-rough hierarchical micro/nano structures to control SW10 cell attachment and migration. It is shown that, depending on the laser processing conditions, distinct cell-philic or cell-repellant patterned areas can be attained with a desired motif. We envisage that our technique could enable spatial patterning of cells in a controllable manner, giving rise to advanced capabilities in cell biology research.

Controlling the morphology and outgrowth of nerve and neuroglial cells: The effect of surface topography.

Unlike other tissue types, like epithelial tissue, which consist of cells with a much more homogeneous structure and function, the nervous tissue spans in a complex multilayer environment whose topographical features display a large spectrum of morphologies and size scales. Traditional cell cultures, which are based on two-dimensional cell-adhesive culture dishes or coverslips, are lacking topographical cues and mainly simulate the biochemical microenvironment of the cells. With the emergence of micro- and nano-fabrication techniques new types of cell culture platforms are developed, where the effect of various topographical cues on cellular morphology, proliferation and differentiation can be studied. Different approaches (regarding the material, fabrication technique, topographical characteristics, etc.) have been implemented. The present review paper aims at reviewing the existing body of literature on the use of artificial micro- and nano-topographical features to control neuronal and neuroglial cells' morphology, outgrowth and neural network topology. The cell responses-from phenomenology to investigation of the underlying mechanisms- on the different topographies, including both deterministic and random ones, are summarized. STATEMENT OF SIGNIFICANCE: There is increasing evidence that physical cues, such as topography, can have a significant impact on the neural cell functions. With the aid of micro-and nanofabrication techniques, new types of cell culture platforms are developed and the effect of surface topography on the cells has been studied. The present review article aims at reviewing the existing body of literature reporting on the use of various topographies to study and control the morphology and functions of cells from nervous tissue, i.e. the neuronal and the neuroglial cells. The cell responses-from phenomenology to investigation of the underlying mechanisms- on the different topographies, including both deterministic and random ones, are summarized.

Data in support on the shape of Schwann cells and sympathetic neurons onto microconically structured silicon surfaces.

This article contains data related to the research article entitled "Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth" in the Biomaterials journal [1]. Scanning electron microscopy (SEM) analysis is performed to investigate whether Schwann cells and sympathetic neurons alter their morphology according to the underlying topography, comprising arrays of silicon microcones with anisotropic geometrical characteristics [1]. It is observed that although soma of sympathetic neurons always preserves its round shape, this is not the case for Schwann cells that become highly polarized in high roughness microconical substrates.

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth.

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

Microconical silicon structures influence NGF-induced PC12 cell morphology.

Micro-and nanofabrication techniques provide the opportunity to develop new types of cell culture platform, where the effect of various topographical cues on cellular functions such as proliferation and differentiation can be studied. In this study, PC12 cells were cultured on patterned silicon (Si) surfaces comprising arrays of microcones (MCs) exhibiting different geometrical characteristics and surface chemistries. It was illustrated that, in the absence of nerve growth factor (NGF), PC12 cells increased proliferation on all types of patterned surface, as compared to flat Si surfaces. However, in the presence of NGF, PC12 cells showed different responses, depending on the plating surface. Unlike low and intermediate rough MC surfaces, highly rough ones exhibiting large distances between MCs did not support PC12 cell differentiation, independently of the MCs' chemical coatings. These results suggest that the geometrical characteristics of MCs alone can influence specific cellular functions. Tailoring of the physical properties of arrays of Si MCs in order to identify which combinations of MC topologies and spatially defined chemistries are capable of driving specific cellular responses is envisaged.

Direct laser writing of 3D scaffolds for neural tissue engineering applications.

This study reports on the production of high-resolution 3D structures of polylactide-based materials via multi-photon polymerization and explores their use as neural tissue engineering scaffolds. To achieve this, a liquid polylactide resin was synthesized in house and rendered photocurable via attaching methacrylate groups to the hydroxyl end groups of the small molecular weight prepolymer. This resin cures easily under UV irradiation, using a mercury lamp, and under femtosecond IR irradiation. The results showed that the photocurable polylactide (PLA) resin can be readily structured via direct laser write (DLW) with a femtosecond Ti:sapphire laser and submicrometer structures can be produced. The maximum resolution achieved is 800 nm. Neuroblastoma cells were grown on thin films of the cured PLA material, and cell viability and proliferation assays revealed good biocompatibility of the material. Additionally, PC12 and NG108-15 neuroblastoma growth on bespoke scaffolds was studied in more detail to assess potential applications for neuronal implants of this material.

Biomimetic micro∕nanostructured functional surfaces for microfluidic and tissue engineering applications.

This paper reviews our work on the application of ultrafast pulsed laser micro∕nanoprocessing for the three-dimensional (3D) biomimetic modification of materials surfaces. It is shown that the artificial surfaces obtained by femtosecond-laser processing of Si in reactive gas atmosphere exhibit roughness at both micro- and nanoscales that mimics the hierarchical morphology of natural surfaces. Along with the spatial control of the topology, defining surface chemistry provides materials exhibiting notable wetting characteristics which are potentially useful for open microfluidic applications. Depending on the functional coating deposited on the laser patterned 3D structures, we can achieve artificial surfaces that are (a) of extremely low surface energy, thus water-repellent and self-cleaned, and (b) responsive, i.e., showing the ability to change their surface energy in response to different external stimuli such as light, electric field, and pH. Moreover, the behavior of different kinds of cells cultured on laser engineered substrates of various wettabilities was investigated. Experiments showed that it is possible to preferentially tune cell adhesion and growth through choosing proper combinations of surface topography and chemistry. It is concluded that the laser textured 3D micro∕nano-Si surfaces with controllability of roughness ratio and surface chemistry can advantageously serve as a novel means to elucidate the 3D cell-scaffold interactions for tissue engineering applications.

Tuning cell adhesion by controlling the roughness and wettability of 3D micro/nano silicon structures.

The aim of this study is to investigate fibroblast cell adhesion and viability on highly rough three-dimensional (3D) silicon (Si) surfaces with gradient roughness ratios and wettabilities. Culture surfaces were produced by femtosecond (fs) laser structuring of Si wafers and comprised forests of conical spikes exhibiting controlled dual-scale roughness at both the micro- and the nano-scale. Variable roughness could be achieved by changing the laser pulse fluence and control over wettability and therefore surface energy could be obtained by covering the structures with various conformal coatings, which altered the surface chemistry without, however, affecting morphology. The results showed that optimal cell adhesion was obtained for small roughness ratios, independently of the surface wettability and chemistry, indicating a non-monotonic dependence of fibroblast adhesion on surface energy. Additionally, it was shown that, for the same degree of roughness, a proper change in surface energy could switch the behaviour from cell-phobic to cell-philic and vice versa, transition that was always correlated to surface wettability. These experimental findings are discussed on the basis of previous theoretical models describing the relation of cell response to surface energy. The potential use of the patterned Si substrates as model scaffolds for the systematic exploration of the role of 3D micro/nano morphology and/or surface energy on cell adhesion and growth is envisaged.

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

IFN-gamma facilitates release of class II-loaded intracellular pools in trophoblast cells: a novel property independent of protein synthesis.

Interferon-gamma (IFN-gamma) is an abortion-inducing factor, yet its effects in such a reaction are subject to various levels of regulation. The trophoblast cell line TROPHO-1 can be induced by IFN-gamma to express mRNA and surface class II major histocompatibility complex (MHC) proteins after 8 and 48 h of stimulation, respectively. Untreated cells, however, show an intracellular accumulation of class II antigens earlier (6 h), indicating the existence of MHC pools in the cystosol independent of any induction. On addition of IFN-y, immunofluorescence, subcellular fractionation, and ELISA experiments showed that class II antigen activity detected in the endosomal compartments of the cells could be measured in the culture supernatants. These soluble class II proteins, when isolated and purified using magnetic bead isolation techniques and tested in SDS-PAGE gel and Western blot experiments, had a molecular weight of 70 kDa. Administration of these molecules to pregnant mice as culture supernatants increased the abortion rate and decreased maternal hematocrit levels, effects that could be immunoabsorbed by anti-I-A(d) monoclonal antibodies (mAb). These results indicate that although surface class II molecules are not expressed on trophoblast cells, they accumulate in endosomal compartments and can be released from the cells on addition of IFN-gamma. This new IFN-gamma property, to mobilize intracellular pools of class II MHC antigens in trophoblast cells independent of de novo protein synthesis and induce their release to the extracellular matrix, is a mechanism that appears to be involved in the fetal rejection process, facilitating priming of the maternal organism against the fetal allograft.

Inhibition of nitric oxide production rescues LPS-induced fetal abortion in mice.

In this report, we examined the involvement of the cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 as well as nitric oxide (NO) in the lipopolysaccharide (LPS)-induced experimental abortion model in BALB/c mice. Although in vivo administration of LPS in pregnant mice showed a 72% decrease of serum IL-10, no significant difference in serum TNF-alpha, IFN-gamma, and IL-4 levels, compared to controls, could be detected. At the same time, a correlation of fetal abortion and maternal splenomegaly with an important increase of NO synthesis in the serum was obtained. Simultaneous administration of LPS and aminoguanidine (AG; an inhibitor to NO synthase) rescued the LPS-induced fetal abortion, reduced maternal spleen weight to physiological levels, and decreased serum NO concentration to control levels. In vitro experiments showed that LPS directly induced NO production in primary placental cells and the TPOPHO-1 trophoblast cell line by stimulating the inducible isoform of NO synthase, which ultimately could be blocked by the NO synthase inhibitors AG and L-NAME. The results indicate that LPS, despite its beneficial involvement in intracellular infections, participates in inflammatory/autoimmune damage during pregnancy, leading to embryotoxicity, which is closely linked to the NO pathway.

Serum levels of pro- and anti-inflammatory cytokines in non-pregnant women, during pregnancy, labour and abortion.

Disturbance of the cytokine equilibrium has been accused for many pathological disorders. Microbial infections, autoimmune diseases, graft rejection have been correlated to over- or under-production of specific cytokines which are produced as responder molecules to the various immune stimuli. The sole naturally occurring immune reaction in the organism is developed during the gestational period where, despite the presence of a semi-allogeneic graft, maternal immunoreactivity is driven to support fetal growth. The successful embryo development has been attributed to the important intervention of cytokines where some have been characterized as indispensable and others deleterious to fetal growth. However, the physiological levels of many factors during the gestational process have not been determined. Thus, in the present study we have measured and established the values of IL-1alpha, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, GM-CSF, TNF-alpha and IFN-gamma during all phases of human pregnancy (first, second and third trimester of pregnancy, labour, abortions of the first trimester) as well as in the non-pregnant control state. This is an attempt to assess serum protein concentrations and present the physiological levels of these cytokines at certain time intervals providing thus a diagnostic advantage in pregnancy cases where the mother cannot immunologically support the fetus. Exploitation of this knowledge and further research may be useful for therapeutic interventions in the future.

Production of embryotoxic IgG antibodies during IFN-gamma treatment of pregnant mice.

PROBLEM: Administration of IFN-gamma during pregnancy in mice is deleterious not only to fetal survival but also to maternal physiology. Thus, injection of recombinant IFN-gamma from days 6-11 of gestation results in significant increase of fetal abortion, decrease of fetal weight accompanied by morphological defects of the embryo, and induction of class II MHC antigens on the spongiotrophoblast zone of the placenta. At the maternal level, this treatment causes splenomegaly, decrease of hematocrit levels, and increase of IgG production. In an attempt to dissect out the different phenomena observed, we examined the properties of polyclonal IgG antibodies contained in the animals' serum as to their ability to recognize antigenic determinants on IFN-gamma-induced placentae and isolated trophoblasts. METHOD: Serum from IFN-gamma-treated pregnant mice was tested in vitro for its ability to recognize specific structures on primary trophoblasts and placental sections induced by IFN-gamma. In vivo this serum was injected in pregnant mice, and the outcome of pregnancy was evaluated. Monoclonal antibodies, resulting from the fusion of spleen cells from IFN-gamma-treated pregnant mice to a myeloma cell line, were used to certify the IgG-dependent embryotoxic effects observed with the polyclonal serum. RESULTS: It was demonstrated that both the polyclonal serum and the monoclonal antibodies recognize antigenic determinants only on the IFN-gamma-induced trophoblasts, placentae, and embryos, reduce fetal size, and cause splenomegaly in the mother, but do not affect the percentage of abortions as compared to controls. CONCLUSIONS: IFN-gamma-induces specific protein(s) on trophoblasts, which are responsible for embryotoxic antibody production in the mother. Since human abortion has been correlated with the production of embryotoxic IgG antibodies, this animal model may prove to be a useful tool in the analysis of events leading to pregnancy loss.