Anastasia Koroleva

Laser Fabrication of Large-Scale Nanoparticle Arrays for Sensing Applications

A novel method for high-speed fabrication of large scale periodic arrays of nanoparticles (diameters 40-200 nm) is developed. This method is based on a combination of nanosphere lithography and laser-induced transfer. Fabricated spherical nanoparticles are partially embedded into a polymer substrate. They are arranged into a hexagonal array and can be used for sensing applications. An optical sensor with the sensitivity of 365 nm/RIU and the figure of merit of 21.5 in the visible spectral range is demonstrated.

Fabrication of microscale medical devices by two-photon polymerization with multiple foci via a spatial light modulator

Two-photon polymerization is an appealing technique for producing microscale devices due to its flexibility in producing structures with a wide range of geometries as well as its compatibility with materials suitable for biomedical applications. The greatest limiting factor in widespread use of two-photon polymerization is the slow fabrication times associated with line-by-line, high-resolution structuring. In this study, a recently developed technology was used to produce microstructures by two-photon polymerization with multiple foci, which significantly reduces the production time. Computer generated hologram pattern technology was used to generate multiple laser beams in controlled positions from a single laser. These multiple beams were then used to simultaneously produce multiple microstructures by two-photon polymerization. Arrays of micro-Venus structures, tissue engineering scaffolds, and microneedle arrays were produced by multifocus two-photon polymerization. To our knowledge, this work is the first demonstration of multifocus two-photon polymerization technology for production of a functional medical device. Multibeam fabrication has the potential to greatly improve the efficiency of two-photon polymerization production of microscale devices such as tissue engineering scaffolds and microneedle arrays.

Two-photon polymerization-generated and micromolding-replicated 3D scaffolds for peripheral neural tissue engineering applications

In this study, we explore the production of well-defined macroscopic scaffolds with two-photon polymerization (2PP) and their use as neural tissue engineering scaffolds. We also demonstrate that these 3D scaffolds can be replicated via soft lithography, which increases production efficiency. Photopolymerizable polylactic acid (PLA) was used to produce scaffolds by 2PP and soft lithography. We assessed the biocompatibility of these scaffolds using an SH-SY5Y human neuronal cell line and primary cultured rat Schwann cells (of direct relevance to the repair of nerve injuries). A Comet assay with SH-SY5Y human neuronal cells revealed minimal DNA damage after washing the photocured material for 7 days in ethanol. Additionally, thin films and 3D scaffolds of the photocured PLA sustained a high degree of Schwann cell purity (99%), enabled proliferation over 7 days and provided a suitable substrate for supporting Schwann cell adhesion such that bi-polar and tri-polar morphologies were observed. Evidence of orthogonally aligned and organized actin thin filaments and the formation of focal contacts were observed for the majority of Schwann cells. In summary, this work supports the use of PLA as a suitable material for supporting Schwann cell growth and in turn use of 3D soft lithography for the synthesis of neural scaffolds in nerve repair.

Fabrication of fibrin scaffolds with controlled microscale architecture by a two-photon polymerization?micromolding technique

Fabrication of three-dimensional (3D) fibrin scaffolds with tightly controllable pore sizes and interconnections has been investigated. The scaffolds were produced using a combination of two-photon polymerization (2PP) and micromolding techniques. Master structures were fabricated by 2PP and regenerated in fibrin by a two-step microreplication procedure. Scanning electron and optical microscopy observations showed that the fibrin scaffolds exhibited a highly porous and interconnected structure. Seeding of endothelial cells in fibrin scaffolds resulted in their directed lining and spreading within network of microreplicated pores, whereas encapsulation of endothelial cells in fibrin gel blocks led to their chaotic and irregular distribution within constructs. These results demonstrate that the 2PP-micromolding technique is suitable for fabrication of complex 3D structures from natural proteins for tissue engineering applications.

Two-photon polymerization of polyethylene glycol diacrylate scaffolds with riboflavin and triethanolamine used as a water-soluble photoinitiator

AIM In this study, the suitability of a mixture containing riboflavin (vitamin B2) and triethanolamine (TEOHA) as a novel biocompatible photoinitiator for two-photon polymerization (2PP) processing was investigated. MATERIALS & METHODS Polyethylene glycol diacrylate was crosslinked using Irgacure(®) 369, Irgacure 2959 or a riboflavin-TEOHA mixture; biocompatibility of the photopolymer extract solutions was subsequently assessed via endothelial cell proliferation assay, endothelial cell viability assay and single-cell gel electrophoresis (comet) assay. Use of a riboflavin-TEOHA mixture as a photoinitiator for 2PP processing of a tissue engineering scaffold and subsequent seeding of this scaffold with GM-7373 bovine aortic endothelial cells was also demonstrated. RESULTS The riboflavin-TEOHA mixture was found to produce much more biocompatible scaffolds than those produced with Irgacure 369 or Irgacure 2959. CONCLUSION The results suggest that riboflavin is a promising component of photoinitiators for 2PP fabrication of tissue engineering scaffolds and other medically relevant structures (e.g., biomicroelectromechanical systems).

Osteogenic differentiation of human mesenchymal stem cells in 3-D Zr-Si organic-inorganic scaffolds produced by two-photon polymerization technique.

Two-photon polymerization (2PP) is applied for the fabrication of 3-D Zr-Si scaffolds for bone tissue engineering. Zr-Si scaffolds with 150, 200, and 250 μm pore sizes are seeded with human bone marrow stem cells (hBMSCs) and human adipose tissue derived stem cells (hASCs) and cultured in osteoinductive and control media for three weeks. Osteogenic differentiation of hASCs and hBMSCs and formation of bone matrix is comparatively analyzed via alkaline phosphatase activity (ALP), calcium quantification, osteocalcin staining and scanning electron microscopy (SEM). It is observed that the 150 μm pore size Zr-Si scaffolds support the strongest matrix mineralization, as confirmed by calcium deposition. Analysis of ALP activity, osteocalcin staining and SEM observations of matrix mineralization reveal that mesenchymal stem cells cultured on 3-D scaffolds without osteogenic stimulation spontaneously differentiate towards osteogenic lineage. Nanoindentation measurements show that aging of the 2PP-produced Zr-Si scaffolds in aqueous or alcohol media results in an increase in the scaffold Young’s modulus and hardness. Moreover, accelerated formation of bone matrix by hASCs is noted, when cultured on the scaffolds with lower Young’s moduli and hardness values (non aged scaffolds) compared to the cells cultured on scaffolds with higher Young’s modulus and hardness values (aged scaffolds). Presented results support the potential application of Zr-Si scaffolds for autologous bone tissue engineering.

3D in vitro platform produced by two-photon polymerization for the analysis of neural network formation and function

Zr-Si organic–inorganic scaffolds fabricated by a two-photon polymerization technique were used for the primary culture of mouse embryonic neural cells. We observed that dissociated hippocampal cells adhere to the scaffolds, produce neurites, elongate and differentiate into adult neurons. Neuronal outgrowth and synaptogenesis were confirmed by immunohistochemical staining with antibodies against βIII-Tubulin and synaptophysin. The formation of a functional neural network was assessed by the measurement of spontaneous activity using Ca2+ imaging of dissociated hippocampal cultures grown on Zr-Si scaffolds. The results of this study suggest that two-photon-induced polymerization of organic–inorganic hybrid biomaterials provides a robust model for 3D neuronal tissue engineering studies.

Two-photon polymerization of 3-D zirconium oxide hybrid scaffolds for long-term stem cell growth

Two-photon polymerization is a technique that involves simultaneous absorption of two photons from a femtosecond laser for selective polymerization of a photosensitive material. In this study, two-photon polymerization was used for layer-by-layer fabrication of 3-D scaffolds composed of an inorganic-organic zirconium oxide hybrid material. Four types of scaffold microarchitectures were created, which exhibit layers of parallel line features at various orientations as well as pores between the line features. Long-term cell culture studies involving human bone marrow stromal cells were conducted using these 3-D scaffolds. Cellular adhesion and proliferation were demonstrated on all of the scaffold types; tissuelike structure was shown to span the pores. This study indicates that two-photon polymerization may be used to create microstructured scaffolds out of an inorganic-organic zirconium oxide hybrid material for use in 3-D tissue culture systems.

Compatibility of cells of the nervous system with structured biodegradable chitosan-based hydrogel matrices

Hydrogel matrices for cell cultivation have been generated by two-photon laser polymerization of unsaturated chitosan derivatives and methacrylated hyaluronic acid. The adhesive and toxic properties of the matrices have been assessed, and the matrices have been shown to have a good compatibility with primary hippocampal cell cultures. The formation of morphologically normal neural networks by cells of the nervous system cultured on the surface of hydrogel matrices has been observed. The metabolic status of dissociated hippocampal cells cultured on the matrices was similar to that of the control cultures, as shown by the results of MTT reductase activity assay. Thus, matrices based on unsaturated polysaccharide derivatives crosslinked by laser irradiation showed good compatibility with differentiated cells of the nervous system and considerable potential for use in neurotransplantation.

Laser microstructured biodegradable scaffolds

Abstract The two-photon polymerization technique (2PP) uses non-linear absorption of femtosecond laser pulses to selectively polymerize photosensitive materials. 2PP has the ability to fabricate structures with a resolution from tens of micrometers down to hundreds of nanometers. Three-dimensional microstructuring by the 2PP technique provides many interesting possibilities for biomedical applications. This microstructuring technique is suitable with many biocompatible polymeric materials, such as polyethylene glycol, polylactic acid, polycaprolactone, gelatin, zirconium-based hybrids, and others. The process of fabrication does not require clean room conditions and does not use hazard chemicals or high temperatures. The most beneficial property of 2PP is that it is capable of producing especially complex three-dimensional (3-D) structures, including devices with overhangs, without using any supportive structure. The flexibility in controlling geometries and feature sizes and the possibility to fabricate structures without the addition of new material layers makes this technique particularly appealing for fabrication of 3-D scaffolds for tissue engineering.