Development of an Oriented Co-Culture System Using 3D Scaffolds Fabricated via Non-Linear Lithography.

Damage in the Peripheral Nervous System (PNS) is related to numerous neurodegenerative diseases and has consequently drawn the attention of Tissue Engineering (TE), which is considered a promising alternative to already established methods such as surgery and autografts. TE focuses on the design, optimization, and use of scaffolds in vitro and in vivo. In this work, the authors used a novel scaffold geometry fabricated via Multiphoton Lithography (MPL), a commonly used fabrication method, for the mono- and co-cultures of glial Schwann (SW10) and neuronal Neuro-2a (N2a) cells. Both cell types have already been used for the study of various neurodegenerative diseases. However, their focus has been on only one of the cell types at a time, with studies regarding their co-culture only recently documented. Here, the suitability of the fabricated scaffolds has been explored and the effects of topography on SW10 and N2a behavior have been investigated. Our findings demonstrate that scaffold co-culture systems favor the presence of neurites compared to mono-cultures at 21 days (31.4 ± 5.5% and 15.4 ± 5.4%, respectively), while there is also a significant decrease in long neurites in the mono-culture over time (45.3 ± 15.9% at 7 days versus 15.4 ± 5.4% at 21 days). It has been shown that the scaffolds can successfully manipulate cell growth, elongation, and morphology, and these results can form a basis for the development of an experimental model for the study of PNS-related diseases and understanding of key cell functions such as myelination.

Strength through defects: A novel Bayesian approach for the optimization of architected materials.

We use a previously unexplored Bayesian optimization framework, "evolutionary Monte Carlo sampling," to systematically design the arrangement of defects in an architected microlattice to maximize its strain energy density before undergoing catastrophic failure. Our algorithm searches a design space with billions of 4 × 4 × 5 3D lattices, yet it finds the global optimum with only 250 cost function evaluations. Our optimum has a normalized strain energy density 12,464 times greater than its commonly studied defect-free counterpart. Traditional optimization is inefficient for this microlattice because (i) the design space has discrete, qualitative parameter states as input variables, (ii) the cost function is computationally expensive, and (iii) the design space is large. Our proposed framework is useful for architected materials and for many optimization problems in science and elucidates how defects can enhance the mechanical performance of architected materials.

Design and Characterization of Microscale Auxetic and Anisotropic Structures Fabricated by Multiphoton Lithography.

The need for control of the elastic properties of architected materials has been accentuated due to the advances in modelling and characterization. Among the plethora of unconventional mechanical responses, controlled anisotropy and auxeticity have been promulgated as a new avenue in bioengineering applications. This paper aims to delineate the mechanical performance of characteristic auxetic and anisotropic designs fabricated by multiphoton lithography. Through finite element analysis the distinct responses of representative topologies are conveyed. In addition, nanoindentation experiments observed in-situ through scanning electron microscopy enable the validation of the modeling and the observation of the anisotropic or auxetic phenomena. Our results herald how these categories of architected materials can be investigated at the microscale.

Split-cube-resonator-based metamaterials for polarization-selective asymmetric perfect absorption.

A split-cube-resonator-based metamaterial structure that can act as a polarization- and direction-selective perfect absorber for the infrared region is theoretically and experimentally demonstrated. The structure, fabricated by direct laser writing and electroless silver plating, is comprised of four layers of conductively-coupled split-cube magnetic resonators, appropriately rotated to each other to bestow the desired electromagnetic properties. We show narrowband polarization-selective perfect absorption when the structure is illuminated from one side; the situation is reversed when illuminating from the other side, with the orthogonal linear polarization being absorbed. The absorption peak can be tuned in a wide frequency range by a sparser or denser arrangement of the split cube resonators, allowing to cover the entire atmospheric transparency window. The proposed metamaterial structure can find applications in polarization-selective thermal emission at the IR atmospheric transparency window for radiative cooling, in cost-effective infrared sensing devices, and in narrowband filters and linear polarizers in reflection mode.

Additive Manufacturing: Applications and Directions in Photonics and Optoelectronics.

The combination of materials with targeted optical properties and of complex, 3D architectures, which can be nowadays obtained by additive manufacturing, opens unprecedented opportunities for developing new integrated systems in photonics and optoelectronics. The recent progress in additive technologies for processing optical materials is here presented, with emphasis on accessible geometries, achievable spatial resolution, and requirements for printable optical materials. Relevant examples of photonic and optoelectronic devices fabricated by 3D printing are shown, which include light-emitting diodes, lasers, waveguides, optical sensors, photonic crystals and metamaterials, and micro-optical components. The potential of additive manufacturing applied to photonics and optoelectronics is enormous, and the field is still in its infancy. Future directions for research include the development of fully printable optical and architected materials, of effective and versatile platforms for multimaterial processing, and of high-throughput 3D printing technologies that can concomitantly reach high resolution and large working volumes.

Multiphoton 3D Printing of Biopolymer-Based Hydrogels.

Multiphoton lithography, based on multiphoton polymerization, is a powerful technique for the fabrication of complex three-dimensional (3D) structures. Herein, we report on the photostructuring of novel biopolymer-based hybrid hydrogels, comprising gelatin methacrylamide and a water-soluble chitosan derivative, via multiphoton polymerization. The nontoxic, Food and Drug Administration-approved, biocompatible photosensitizer eosin Y was exploited as the sole photoinitiator, without the coinitiators and/or comonomer that are commonly used, allowing for further expansion of the available wavelengths up to 800 nm. Importantly, the obtained hybrid material exhibits excellent biocompatibility, evidenced by the increased proliferation of dental pulp stem cells, compared with the individual components and the polystyrene control, after 7 days in culture. Additionally, the 3D hybrid scaffolds promote the matrix mineralization, following their functionalization with bone morphogenetic protein 2. These tailor-made synthetic, biocompatible materials pave the way for further opportunities in 3D scaffold fabrication, including in situ and in vivo biofabrication.

Quantum dot based 3D printed woodpile photonic crystals tuned for the visible.

The development of dynamically responsive 3D photonic elements, which is crucial for the design of active integrated photonic circuits, requires the incorporation of material systems with fast and tunable response. To this end, semiconductor quantum dots have been widely used to perform as the active material system to be integrated; nonetheless, multiple-step processing is usually required for the active functions to be preserved, thereby restricting functionality of integrated 3D quantum photonic elements mostly to the infrared. Here, we report a simple scheme for the realization of visible light active 3D photonic devices by combining direct laser writing with two-photon absorption and in situ synthesis of cadmium sulfide (CdS) nanoparticles. The novel active 3D printable hybrid material is synthesized by crosslinking precursors of CdS quantum dots into a photo-structurable organic-inorganic zirconium-silicon hybrid composite integrating functional properties of both high spatial resolution and high third-order nonlinearity into the photonic matrix. As a proof-of-demonstration for 3D printed active photonic devices, woodpile photonic crystals with an inlayer periodicity down to 500 nm are successfully fabricated showing clear photonic stop bands in the visible spectral region, while for the first time, evidence of an ultrafast dynamic response in the visible is also demonstrated.

Guided Assembly of Block Copolymers in Three-Dimensional Woodpile Scaffolds.

Three-dimensional (3D) nanofabrication using the directed self-assembly of block copolymers (BCPs) holds great promise for the nanoscale device fabrication and integration into 3D architectures over large areas with high element densities. In this work, a robust platform is developed for building 3D BCP architectures with tailored functionality using 3D micron-scale woodpile structures (WPSs), fabricated by a multiphoton polymerization technique. By completely filling the spaces of the WPSs and using the interactions of the blocks of the BCPs with the struts of the WPS, well-developed 3D nanoscopic morphologies are produced. Metal ion complexation with one block of the copolymer affords a convenient stain to highlight one of the microdomains of the copolymer for electron microscopy studies but also, with the reduction of the complexing salt to the corresponding metal, a simple strategy is shown to produce 3D constructs with nanoscopic domain resolution.

3D laser nano-printing on fibre paves the way for super-focusing of multimode laser radiation.

Multimode high-power laser diodes suffer from inefficient beam focusing, leading to a focal spot 10-100 times greater than the diffraction limit. This inevitably restricts their wider use in 'direct-diode' applications in materials processing and biomedical photonics. We report here a 'super-focusing' characteristic for laser diodes, where the exploitation of self-interference of modes enables a significant reduction of the focal spot size. This is achieved by employing a conical microlens fabricated on the tip of a multimode optical fibre using 3D laser nano-printing (also known as multi-photon lithography). When refracted by the conical surface, the modes of the fibre-coupled laser beam self-interfere and form an elongated narrow focus, usually referred to as a 'needle' beam. The multiphoton lithography technique allows the realisation of almost any optical element on a fibre tip, thus providing the most suitable interface for free-space applications of multimode fibre-delivered laser beams. In addition, we demonstrate the optical trapping of microscopic objects with a super-focused multimode laser diode beam thus rising new opportunities within the applications sector where lab-on-chip configurations can be exploited. Most importantly, the demonstrated super-focusing approach opens up new avenues for the 'direct-diode' applications in material processing and 3D printing, where both high power and tight focusing is required.

The role of intramolecular charge transfer and symmetry breaking in the photophysics of pyrrolo[3,2-b]pyrrole-dione.

A three-step synthetic route to a structurally unique π-expanded pyrrolo[3,2-b]pyrrole derived bis-ketone has been developed. In contrast to all previous ladder-type pyrrolopyrroles, the new dye exhibits a low-energy absorption band in the visible region which is responsible for its red-purple color. Interestingly, even though the compound is centrosymmetric, this band coincides with the lowest energy two-photon absorption (TPA) transition. This non-typical behaviour has been computationally rationalized by finding two close lying excited states, one of which (S1) is active for OPA and the other (S2) for TPA processes, which arise from the mixing of two symmetric partial charge-transfer states. The ultrafast excited-state dynamics was characterized by means of transient absorption analysis. A relaxation process involving S1 symmetry breaking occurs in a few ps, leading to the formation of the lowest energy charge-transfer state. This is weakly emitting, with a measured lifetime in the order of tens of picoseconds. Interestingly, two-photon polymerization has been achieved using this new ketone. The high yield of radical photo-initiation upon two-photon excitation was demonstrated by the fabrication of woodpile photonic crystal templates by direct laser writing using a zirconium-silicon hybrid composite.

Ring-Airy beams at the wavelength limit.

We demonstrate that paraxial ring-Airy beams can approach the wavelength limit, while observing a counterintuitive, strong enhancement of their focal peak intensity. Using numerical simulations, we show that this behavior is a result of the coherent constructive action of paraxial and nonparaxial energy flow. A simple theoretical model enables us to predict the parameter range over which this is possible.

3D micro-structured arrays of ZnΟ nanorods.

The fabrication of nanostructures with controlled assembly and architecture is very important for the development of novel nanomaterial-based devices. We demonstrate that laser techniques coupled with low-temperature hydrothermal growth enable complex three-dimensional ZnO nanorod patterning on various types of substrates and geometries. This methodology is based on a procedure involving the 3D scaffold fabrication using Multi-Photon Lithography of a photosensitive material, followed by Zn seeded Aqueous Chemical Growth of ZnO nanorods. 3D, uniformly aligned ZnO nanorods are produced. The increase in active surface area, up to 4.4 times in the cases presented here, provides a dramatic increase in photocatalytic performance, while other applications are also proposed.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) immobilized on laser-fabricated 3D scaffolds enhance osteogenesis.

The regeneration of bone via a tissue engineering approach involves components from the macroscopic to the nanoscopic level, including appropriate 3D scaffolds, cells and growth factors. In this study, hexagonal scaffolds of different diagonals were fabricated by Direct Laser Writing using a photopolymerizable hybrid material. The proliferation of bone marrow (BM) mesenchymal stem cells (MSCs) cultured on structures with various diagonals, 50, 100, 150 and 200µm increased significantly after 10days in culture, however without significant differences among them. Next, recombinant human bone morphogenetic protein 2 (rhBMP-2) was immobilized onto the hybrid material both via covalent binding and physical adsorption. Both immobilization types exhibited similar high releaseate bioactivity profiles and a sustained delivery of rhBMP-2. The collagen and calcium levels produced in the extracellular matrix (ECM) were significantly elevated for the samples functionalized with BMP-2 compared to those in the osteogenic medium. Furthermore, significant upregulation of gene expression in both types of BMP-2 immobilized scaffolds was observed for alkaline phosphatase (ALPL) and osteocalcin (BGLAP) at days 7, 14, and 21, for RUNX2 at day 21, and for osteonectin (SPARC) at days 7 and 14. The results suggest that the release of bioactive rhBMP-2 from the hybrid scaffolds enhance the control over the osteogenic differentiation during cell culture.

3D plasmonic crystal metamaterials for ultra-sensitive biosensing.

We explore the excitation of plasmons in 3D plasmon crystal metamaterials and report the observation of a delocalized plasmon mode, which provides extremely high spectral sensitivity (>2600 nm per refractive index unit (RIU) change), outperforming all plasmonic counterparts excited in 2D nanoscale geometries, as well as a prominent phase-sensitive response (>3*10(4) deg. of phase per RIU). Combined with a large surface for bioimmobilization provided by the 3D matrix, the proposed sensor architecture promises a new important landmark in the advancement of plasmonic biosensing technology.

Burr-like, laser-made 3D microscaffolds for tissue spheroid encagement.

The modeling, fabrication, cell loading, and mechanical and in vitro biological testing of biomimetic, interlockable, laser-made, concentric 3D scaffolds are presented. The scaffolds are made by multiphoton polymerization of an organic-inorganic zirconium silicate. Their mechanical properties are theoretically modeled using finite elements analysis and experimentally measured using a Microsquisher(®). They are subsequently loaded with preosteoblastic cells, which remain live after 24 and 72 h. The interlockable scaffolds have maintained their ability to fuse with tissue spheroids. This work represents a novel technological platform, enabling the rapid, laser-based, in situ 3D tissue biofabrication.

Adhesion and growth of human bone marrow mesenchymal stem cells on precise-geometry 3D organic-inorganic composite scaffolds for bone repair.

Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2-(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50mol% DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide, and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material containing 50% DMAEMA from the first 2h after seeding, and up to several days, and a proliferation increase after 14 and 21days, similar to the polystyrene control, independent of cell donor. These findings support the potential use of our proposed cell-material combination in bone tissue engineering.

Advances in Functional Assemblies for Regenerative Medicine.

The ability to synthesise bioresponsive systems and selectively active biochemistries using polymer-based materials with supramolecular features has led to a surge in research interest directed towards their development as next generation biomaterials for drug delivery, medical device design and tissue engineering.

π-Expanded α,ß-unsaturated ketones: synthesis, optical properties, and two-photon-induced polymerization.

A library of π-expanded α,ß-unsaturated ketones was designed and synthesized. They were prepared by a combination of Wittig reaction, Sonogashira reaction, and aldol condensation. It was further demonstrated that the double aldol condensation can be performed effectively for highly polarized styrene- and diphenylacetylene-derived aldehydes. The strategic placement of two dialkylamino groups at the periphery of D-π-A-π-D molecules resulted in dyes with excellent solubility. These ketones absorb light in the region 400-550 nm. Many of them display strong solvatochromism so that the emission ranges from 530-580 nm in toluene to the near-IR region in benzonitrile. Ketones based on cyclobutanone as central moieties display very high fluorescence quantum yields in nonpolar solvents, which decrease drastically in polar media. Photophysical studies of these new functional dyes revealed that they possess an enhanced two-photon absorption cross section when compared with simpler ketone derivatives. Due to strong polarization of the resulting dyes, values of two-photon absorption cross sections on the level of 200-300 GM at 800 nm were achieved, and thanks to that as well as the presence of the keto group, these new two-photon initiators display excellent performance so that the operating region is 5-75 mW in some cases.

Mineralized self-assembled peptides on 3D laser-made scaffolds: a new route toward 'scaffold on scaffold' hard tissue engineering.

In this study, we propose a new approach to hard tissue regeneration based on the mineralization of 3D scaffolds made using lasers. To this end, we report the rational design of aspartate-containing self-assembling peptides targeted for calcium binding. We further investigate the suitability of these peptides to support cell attachment and proliferation when coupled on a hybrid organic-inorganic structurable material, and evaluate the response of pre-osteoblastic cells on functionalized 3D scaffolds and material surfaces. Our results show that the mineralized peptide, when immobilized on a hybrid photo-structurable material strongly supports cell adhesion, a proliferation increase after three and seven days in culture, and exhibits a statistically significant increase of biomineralization. We propose this strategy as a 'scaffold on scaffold' approach for hard tissue regeneration.

Redox multiphoton polymerization for 3D nanofabrication.

We report for the first time on the redox multiphoton polymerization of an organic-inorganic composite material, in which one of the components, a vanadium metallo-organic complex, initiates the polymerization. The composite employs multiphoton absorption to self-generate radicals by photoinduced reduction of the metal species from vanadium (V) to vanadium (IV). We exploit this material for the fabrication of fully 3D structures by multiphoton polymerization with 200 nm resolution, employing a femtosecond laser operating at 800 nm, in the absence of a photoinitiator. Nonlinear absorption measurements indicate that the use of an 800 nm laser initiates the photopolymerization due to three-photon absorption of the vanadium alkoxide. The laser power required to induce this three-photon polymerization is comparable to what is required for inducing two-photon polymerization in materials using standard two-photon absorbers, most likely due to the high content of vanadium in the final composite (up to 50% mole).