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Multiphoton lithography allows the high resolution, free-form 3D printing of structures such as micro-optical elements and 3D scaffolds for Tissue Engineering. A major obstacle in its application in these fields is material and structure autofluorescence. Existing photoresists promise near zero fluorescence at the expense of poor mechanical properties, and low printing efficiency. Sudan Black B is a molecular quencher used as a dye for biological studies and as means of decreasing the autofluorescence of polymers. In our study, we report the use of Sudan Black B both as a photoinitiator and as a post-fabrication treatment step, using the zirconium silicate SZ2080 for the development of a non-fluorescent composite. We use this material for the 3D printing of micro-optical elements, and meso-scale scaffolds for mesenchymal stem cell cultures. Our results show that the Sudan Black B photosensitive hybrid can be used for the fabrication of high resolution, highly transparent, autofluorescence-free microstructures.

Author(s): Flamourakis, George; Spanos, Ioannis; Vangelatos, Zacharias; Manganas, Phanee; Papadimitriou, Lina; Grigoropoulos, Costas; Ranella, Anthi; Farsari, Maria

The use of ultra-short pulses for nanoengineering of biomaterials opens up possibilities for biological, medical and tissue engineering applications. Structuring the surface of a biomaterial into arrays with micro- and nanoscale features and architectures, defines new roadmaps to innovative engineering of materials. Thin films of novel collagen/elastin composite and gelatin were irradiated by Ti:sapphire fs laser in air at central wavelength 800 nm, with pulse durations in the range of 30 fs. The size and shape as well as morphological forms occurring in the resulted areas of interaction were analyzed as a function of irradiation fluence and number of pulses by atomic force microscopy (AFM). The fs interaction regime allows generation of well defined micro porous surface arrays. In this study we examined a novel composite consisting of collagen and elastin in order to create a biodegradable matrix to serve as a biomimetic surface for cell attachment. Confocal microscopy images of modified zones reveal formation of surface fringe patterns with orientation direction alongside the area of interaction. Outside the crater rim a wave-like topography pattern is observed. Structured, on a nanometer scale, surface array is employed for cell-culture experiments for testing cell’s responses to substrate morphology. Mice fibroblasts migration was monitored after 3 days cultivation period using FESEM. We found that fibroblasts cells tend to migrate and adhere along the laser modified zones. The performed study proved that the immobilized collagen based biofilms suite as a template for successful fibroblasts cell guidance and orientation. Fs laser induced morphological modification of biomimetic materials exhibit direct control over fibroblasts behaviour due to induced change in their wettability state.

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.

Controlling the interactions of light with matter is crucial for the success and scalability for materials processing applications at micro and nano-scales. The use of ultrafast pulsed lasers (i.e. lasers emitting pulses of duration shorter than 10 –12 s) for the micro/nano engineering of biomaterials or materials relevant to biological applications opens up several exciting possibilities in this respect. These possibilities rely on several attractive features of ultrafast laser–matter interaction processes which allow nanoscale spatial resolution, non-thermal and non-destructive engineering to take place. This article presents a review of novel laser-based techniques for the printing and micro- and nano- scale surface modification of materials for biological applications. Emphasis is placed on techniques appropriate for biochip and tissue engineering applications, for which there is an increasing demand over the last years. Besides presenting recent advances achieved by these techniques, this work also delineates existing limitations and highlights emerging possibilities and future prospects in this field.

Peptide-based arrays and patterns are a powerful tool in the study of protein functionality, with a variety of applications such as the study of interactions between peptides and enzymes, proteins and DNA. One of the main and most critical issues is the generation of high density arrays which maintain the biological function of the peptides. In this study, we employ Laser-Induced Forward Transfer for the generation of high density photosensitive biotin arrays on ORMOCER®-coated surfaces. We then generate peptide microarrays, which assemble due to the employment of avidin-biotin and thiol chemistry. These initial results are poised to the construction of larger peptide patterns as scaffolds for the incorporation and display of ligands critical for cell attachment and growth, or for the templating of inorganic materials. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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.

Laser-induced forward transfer has been used for the deposition of photoactive biotin in micron-scale patterns. The process uses a 500 fs pulsed KrF laser beam to transfer small amounts of a liquid solution target as micron-size droplets to a substrate placed parallel and in close proximity to it. The biomolecules remain active after the transfer; this is demonstrated through fluorescence assays. In addition to the laser parameters, those regarding the target composition and the receiving surface for the miniaturization of the transferred patterns have been studied and optimized. Droplets as small as 5 μm have been obtained by reducing the target thickness and transfer energy; by increasing the percentage of glycerol added in the biomolecules solution and by using hydrophobic surfaces as receiving substrates. The influence of the glycerol addition and the hydrophobicity of the receiving surfaces on the activity of the transferred biomolecules have also been studied.

In this study, we propose a photostructuring approach for protein films based on a treatment with nanosecond pulses of a KrF excimer laser. As a model protein we used an amyloid fibril-forming protein. Laser treatment induced a foaming of the sample surface exhibiting an interconnected fibrous mesh with a high degree of control and precision. The surface foaming was well characterized by scanning electron microscopy, atomic force microscopy, laser induced fluorescence and contact angle measurements. The laser irradiated areas of the protein films acquired new morphological and physicochemical properties that could be exploited to fulfill unmet challenges in the tissue engineering field. In this context we subsequently evaluated the response of NIH/3T3 fibroblast cell line on the processed film. Our results show a strong and statistically significant preference for adhesion and proliferation of cells on the irradiated areas compared to the non-irradiated ones. We propose that this strategy can be followed to induce selective cell patterning on protein films at the microscale.

The ability to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. Preliminary results have been obtained on collagen modification by irradiation with single and multiple pulses of femtosecond laser with variable pulse duration. Irradiation of collagen thin film by single pulses of femtosecond duration results in creation of foam layer with micrometer thickness. The structure and thickness of the layer strongly depends on the number of the applied laser pulses. The surface properties of collagen thin films before and after Ti-sapphire irradiation with 800 nm were investigated by means of the technique Field Emission Scanning Electron Microscope (FESEM). Based on the FESEM results, it was possible to identify an energy density range as the ablation threshold for collagen thin films. The laser-induced foam formation was characterized over the intensity range 3 – 4.2x10 11 W/cm 2 . The results of the field emission scanning electron microscopy, showed that by tailoring the laser pulse duration, improved the uniformity of the pore network. Examination of the interaction of ultra-short laser pulses with collagen films is useful for controlling the chemical and microstructural modification of the created foam layer.

Tissue Engineering is defined as the technology aiming to apply the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Its eventual goal is the creation of 3D artificial cell culture scaffolds that mimic the natural extracellular environment features sufficiently, so that cells function in the artificial medium as they would in vivo. Cells in tissue are surrounded by a dynamic cell type-dependent extracellular matrix that provides instructive cues at both the micro- and the nanoscale needed to maintain cell phenotype and behaviour. Cells are thus, inherently responsive to their environment, receptive to micro- and nanoscale features and patterns of chemistry and topography. Lasers are increasingly proving to be promising tools for the controlled and reproducible structuring of biomaterials at micro- and nanoscales. This chapter reviews current approaches for laser based fabrication of biomimetic tissue engineering scaffolds. These include laser processing of natural biomaterials synthesized to achieve certain compositions or properties similar to those of the extracellular matrix as well as novel laser fabrication technologies to achieve structural features on artificial materials mimicking the extracellular matrix morphology on various levels. The chapter concludes with the wealth of arising possibilities, demonstrating the excitement and significance of the laser based biomimetic materials processing for tissue engineering and regeneration.

Ultraviolet laser irradiation using pulses with duration from the nanosecond to the femtosecond range was investigated aiming at the generation of a foam layer on films of the biopolymers chitosan, starch and their blend. We report on the morphological characteristics of the foams obtained upon irradiation and on the accompanying laser induced photochemistry, assessed by on line monitoring of the laser induced fluorescence. We identify the laser conditions (pulse duration) at which foaming is produced and discuss the obtained results in reference to the material properties, particularly extinction coefficient and thermal parameters. This article also reports on successful cell culture on the laser induced foam structure generated in chitosan, as an illustrative example of the possibility of broader use of laser induced biopolymer foaming structures in biology., Funding from Ministry of Science and Innovation of Spain (MICINN) under Project CTQ2010-15680. E.R., M.O. and M.S. gratefully acknowledge Juan de la Cierva, CONSOLIDER CSD2007-00058, and Geomateriales (CAM, S2009/Mat-1629) Programs for contracts. The research leading to these results has received support from the access activities of the EC FP7-Infrastructures-2007 Project “Laserlab-Europe II” (grant agreement no: 212025) operating at the Ultraviolet Laser Facility, IESL-FORTH. Prof. S. Lazare is acknowleged for useful discussions and A. Manousaki for SEM measurements.

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.

We report on the fabrication of high resolution 3D scaffolds of polylactide-based materials using direct laser writing and we explore their use as neural tissue engineering scaffolds.

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.

Materials processing by ultrafast lasers offers several attractive possibilities for micro/nano fabrication applications. Several exciting prospects arise in the context of surface and bulk laser induced modifications. These form the basis for diverse applications, including the development and functionalization of laser engineered surfaces, the laser transfer of biomolecules and the functionalization of 3D structures constructed by multiphoton stereolithography. In particular, two examples will be discussed in the following, namely a new approach for the development of superhydrophobic, self cleaning surfaces and the fabrication of functionalized scaffolds, for tissue engineering applications.

We report on the fabrication of high resolution 3D scaffolds of polylactide-based materials using direct laser writing and we explore their use as neural tissue engineering scaffolds.

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.