Aleksandr Ovsianikov

Ultra-Low Shrinkage Hybrid Photosensitive Material for Two-Photon Polymerization Microfabrication

Investigations into the structuring by two-photon polymerization of a nonshrinking, photosensitive, zirconium sol-gel material are presented. This hybrid material can be photostructured even when it contains up to 30 mol % of zirconium propoxide (ZPO); by varying the materials inorganic content, it is possible to modify and tune its refractive index. The introduction of ZPO significantly increases the photosensitivity of the resulting photopolymer. The fabricated three-dimensional photonic crystal structures demonstrate high resolution and a clear band-stop in the near-IR region. In contrast to common practice, no additional effort is required to precompensate for shrinkage or to improve the structural stability of the fabricated photonic crystals; this, combined with the possibility of tuning this materials optical, mechanical, and chemical properties, makes it suitable for a variety of applications by two-photon polymerization manufacturing.

Fabrication of woodpile structures by two-photon polymerization and investigation of their optical properties

Two-photon polymerization (2PP) is a powerful technique for the fabrication of 3D micro- and submicro-structures. By applying laser powers that are only slightly above the polymerization threshold, 3D structuring of photosensitive materials with a resolution down to 100 nm can be realized. Here we report on woodpile photonic crystal structures fabricated in organic-inorganic hybrid polymers (Ormocers) and investigation of their optical properties. The fabricated crystals possess a photonic band gap in the near infrared spectral region. The polymeric woodpile structures can be used as templates for the fabrication of highly refractive TiO2 replicas. First results in this direction are presented.

Laser Fabrication of Three-Dimensional CAD Scaffolds from Photosensitive Gelatin for Applications in Tissue Engineering

In the present work, 3D CAD scaffolds for tissue engineering applications were developed starting from methacrylamide-modified gelatin (GelMOD) using two-photon polymerization (2PP). The scaffolds were cross-linked employing the biocompatible photoinitiator Irgacure 2959. Because gelatin is derived from collagen (i.e., the main constituent of the ECM), the developed materials mimic the cellular microenvironment from a chemical point of view. In addition, by applying the 2PP technique, structural properties of the cellular microenvironment can also be mimicked. Furthermore, in vitro degradation assays indicated that the enzymatic degradation capability of gelatin is preserved for the methacrylamide-modified derivative. An in depth morphological analysis of the 2PP-fabricated scaffolds demonstrated that the parameters of the CAD model are reproduced with great precision, including the ridge-like surface topography on the order of 1.5 μm. The developed scaffolds showed an excellent stability in culture medium. In a final part of the present work, the suitability of the developed scaffolds for tissue engineering applications was verified. The results indicated that the applied materials are suitable to support porcine mesenchymal stem cell adhesion and subsequent proliferation. Upon applying osteogenic stimulation, the seeded cells differentiated into the anticipated lineage. Energy dispersive X-ray (EDX) analysis showed the induced calcification of the scaffolds. The results clearly indicate that 2PP is capable of manufacturing precisely constructed 3D tissue engineering scaffolds using photosensitive polymers as starting material.

Three-dimensional laser micro- and nano-structuring of acrylated poly(ethylene glycol) materials and evaluation of their cytoxicity for tissue engineering applications.

The natural cell environment is characterized by complex three-dimensional structures, which contain features at multiple length scales. Many in vitro studies of cell behavior in three dimensions rely on the availability of artificial scaffolds with controlled three-dimensional topologies. In this paper, we demonstrate fabrication of three-dimensional scaffolds for tissue engineering out of poly(ethylene glycol) diacrylate (PEGda) materials by means of two-photon polymerization (2PP). This laser nanostructuring approach offers unique possibilities for rapid manufacturing of three-dimensional structures with arbitrary geometries. The spatial resolution dependence on the applied irradiation parameters is investigated for two PEGda formulations, which are characterized by molecular weights of 302 and 742. We demonstrate that minimum feature sizes of 200nm are obtained in both materials. In addition, an extensive study of the cytotoxicity of the material formulations with respect to photoinitiator type and photoinitiator concentration is undertaken. Aqueous extracts from photopolymerized PEGda samples indicate the presence of water-soluble molecules, which are toxic to fibroblasts. It is shown that sample aging in aqueous medium reduces the cytotoxicity of these extracts; this mechanism provides a route for biomedical applications of structures generated by 2PP microfabrication and photopolymerization technologies in general. Finally, a fully biocompatible combination of PEGda and a photoinitiator is identified. Fabrication of reproducible scaffold structures is very important for systematic investigation of cellular processes in three dimensions and for better understanding of in vitro tissue formation. The results of this work suggest that 2PP may be used to polymerize poly(ethylene glycol)-based materials into three-dimensional structures with well-defined geometries that mimic the physical and biological properties of native cell environments.

Laser printing of cells into 3D scaffolds

One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

Two-photon polymerization technique for microfabrication of CAD-designed 3D scaffolds from commercially available photosensitive materials.

We report on recent advances in the fabrication of three‐dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two‐photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer‐designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient‐specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold‐like structures, using the photosensitive organic–inorganic hybrid polymer ORMOCER® (ORganically MOdified CERamics) and epoxy‐based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER® and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR‐17 granulosa cells was not affected by the presence of ORMOCER®. Furthermore, gap junction conductance measurements revealed that ORMOCER® did not alter the formation of cell–cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties. Copyright

Bioink properties before, during and after 3D bioprinting

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.

Engineering 3D cell-culture matrices: multiphoton processing technologies for biological and tissue engineering applications

Cells respond to topographical, mechanical and biochemical characteristics of the surrounding environment. Capability to reconstruct these factors individually, and also acting in accord, would facilitate systematic investigations of a multitude of related biological and tissue engineering questions. The subject of the present review is a group of technologies allowing realization of customized cell-culture matrices. These methods utilize photochemistry induced by multiphoton absorption and are carried out using essentially identical equipment. Fabrication of 2D microstructured substrates, complex 3D scaffolds, containing actively induced topographies, and immobilization of biomolecules in a spatially defined manner was demonstrated with these techniques. The reviewed reports indicate that multiphoton processing is a promising technology platform for the development of standard biomimetic microenvironments for 3D cell culture.

Shrinkage of microstructures produced by two-photon polymerization of Zr-based hybrid photosensitive materials

An investigation of the shrinking behaviour of a zirconium-based sol-gel composite micro-structured by two-photon polymerization is presented and a simple, straightforward methodology allowing the evaluation of shrinkage is suggested. It is shown that volume reduction is directly related to the average laser power (irradiation dose) used for the microfabrication and becomes a critical issue near the polymerization threshold. It is demonstrated that this shrinkage can be employed beneficially to improve the structural resolution. This is demonstrated by the presence of stopbands in the photonic crystal nanostructures fabricated with controlled volume reduction. Well above the polymerization threshold, the studied material exhibits remarkably low shrinkage. Therefore, no additional effort for the pre-compensation of distortion and for the improvement of structural stability is required.

Three-dimensional cell growth on structures fabricated from ORMOCER® by two-photon polymerization technique

Two-photon polymerization technique was applied to generate three-dimensional (3D) scaffold-like structures using the photosensitive organic—inorganic hybrid polymer ORMOCER®. The structures were studied with respect to potential applications as scaffold for tissue engineering. Cell counting and comet assay, respectively, demonstrated that doubling time and DNA strand breaks of CHO cells, GFSHR-17 granulosa cells, GM-7373 endothelial cells, and SH-SY5Y neuroblastoma cells were not affected by ORMOCER®. ORMOCER® related alteration of formation of tissue specific cell-to-cell adhesions like gap junctions was ruled out by double whole-cell patch-clamp technique. Additionally, growth of cells on the vertical surfaces of 3D structures composed of ORMOCER® is shown.