Zhiquan Li

Laser Photofabrication of Cell-Containing Hydrogel Constructs

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.

Photo-sensitive hydrogels for three-dimensional laser microfabrication in the presence of whole organisms

Abstract. Hydrogels are polymeric materials with water contents similar to that of soft tissues. Due to their biomimetic properties, they have been extensively used in various biomedical applications including cell encapsulation for tissue engineering. The utilization of photopolymers provides a possibility for the temporal and spatial controlling of hydrogel cross-links. We produced three-dimensional (3-D) hydrogel scaffolds by means of the two-photon polymerization (2PP) technique. Using a highly efficient water-soluble initiator, photopolymers with up to 80 wt.% water were processed with high precision and reproducibility at a writing speed of 10  mm/s. The biocompatibility of the applied materials was verified using Caenorhabditis elegans as living test organisms. Furthermore, these living organisms were successfully embedded within a 200×200×35 μm3 hydrogel scaffold. As most biologic tissues exhibit a window of transparency at the wavelength of the applied femtosecond laser, it is suggested that 2PP is promising for an in situ approach. Our results demonstrate the feasibility of and potential for bio-fabricating 3-D tissue constructs in the micrometre-range via near-infrared lasers in direct contact with a living organism.

Initiation efficiency and cytotoxicity of novel water-soluble two-photon photoinitiators for direct 3D microfabrication of hydrogels

The lack of efficient water-soluble two-photon absorption (TPA) photoinitiators has been a critical obstruction for three dimensional hydrogel microfabrications with high water load by two-photon induced polymerization (TPIP). In this paper, a series of cyclic benzylidene ketone-based two-photon initiators, containing carboxylic acid sodium salts to improve water solubility, were synthesized via classical aldol condensation reactions. The cytotoxicity of cyclopentanone-based photoinitiators is as low as that of the well-known biocompatible photoinitiator Irgacure 2959 as assessed in the dark with MG63 cell line. In z-scan measurement, the TPA cross sections of the investigated initiators are only moderate in water, while the TPA values for hydrophobic analogues measured in chloroform were much higher. All novel initiators exhibited broad processing windows in TPIP tests using hydrophilic photopolymers with up to 50 wt% of water. Impressively, microfabrication of hydrogels with excellent precision was possible at a writing speed as high as 100 mm s−1.

Direct Visualization of Excited-State Symmetry Breaking Using Ultrafast Time-Resolved Infrared Spectroscopy

Most symmetric quadrupolar molecules designed for two-photon absorption behave as dipolar molecules in the S1 electronic excited state. This is usually explained by a breakup of the symmetry in the excited state. However, the origin of this process and its dynamics are still not fully understood. Here, excited-state symmetry breaking in a quadrupolar molecule with a D-π-A-π-D motif, where D and A are electron donating and accepting units, is observed in real time using ultrafast transient infrared absorption spectroscopy. The nature of the relaxed S1 state was found to strongly depend on the solvent polarity: (1) in nonpolar solvents, it is symmetric and quadrupolar; (2) in weakly polar media, the quadrupolar state observed directly after excitation transforms to a symmetry broken S1 state with one arm bearing more excitation than the other; and (3) in highly polar solvents, the excited state evolves further to a purely dipolar S1 state with the excitation localized entirely on one arm. The time scales associated with the transitions between these states coincide with those of solvation dynamics, indicating that symmetry breaking is governed by solvent fluctuations.

Evaluation of 3D structures fabricated with two-photon-photopolymerization by using FTIR spectroscopy

Two-photon-induced photopolymerization (2PP) has gained increased interest due to the capability of manufacturing three-dimensional structures with very high feature resolution. To assess the suitability of photopolymer systems for 2PP, methods have to be developed that allow a screening of the efficiency of monomer-initiator combinations in the context of high throughput, large processing window and geometric quality of the final parts. In this paper, a method for evaluating 2PP structures is described. For this purpose, the double-bond conversion of fabricated 2PP structures was measured giving quantifiable results about the efficiency of the photoinitiator. The method is based on local measurement of the double-bond conversion of the photopolymer using a microscope in combination with infrared spectroscopy. The obtained double-bond conversion is a measure for the efficiency of the photopolymer system (initiator in combination with monomer), and thus allows to compare different photopolymers in a quanti...

Young’s modulus measurement of two-photon polymerized micro-cantilevers by using nanoindentation equipment

Due to its high feature resolution and the capability to produce 3D-structures without the need of support-structures, two-photon polymerization (2PP) is one of the fastest growing technologies in the field of additive manufacturing. To ensure good quality of the fabricated parts, proper methods for evaluating the suitability of different material systems for 2PP are necessary. In this paper, we present a fast screening method based on measuring the Youngs modulus of micro-cantilevers giving a quantifiable measure and representing the material- and fabrication-properties in one experiment.

Two-photon-induced thiol-ene polymerization as a fabrication tool for flexible optical waveguides

In this contribution, we present two flexible thiol-ene-based hybrid materials based on epoxy and acetoxy polysiloxane matrix materials. The latter cross-linking mechanisms allow for orthogonal curing of the matrix in the presence of thiol-ene monomers enabling fast one-step access to two-photon-polymerization (2PP) curable substrates for waveguide fabrication. Another time-saving feature of our concept is the straightforward UV-flood-curing after 2PP, which is also a progress compared to previous works with elaborate postprocessing. Optimization of the ratio of thiol/ene moieties with respect to reactivity and analyses of the thermal stability of the materials, which is required for the industrial process, were carried out. Besides investigations regarding the refractive index of the materials, the proof of principle for successful waveguiding will be given. Flexible optical waveguides were successfully fabricated inside a low refractive polysiloxane matrix material.