Xiaohua Qin

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.

Enzymatic synthesis of hyaluronic acid vinyl esters for two-photon microfabrication of biocompatible and biodegradable hydrogel constructs

Two-photon polymerization (2PP) allows 3D microfabrication of biomaterial scaffolds with user-defined geometry. This technique is highly promising for 3D cell culture and tissue engineering. However, biological applications of 2PP require photopolymerizable hydrogels with high reactivity and low cytotoxicity. This paper describes a novel hydrogel system based on hyaluronic acid vinyl esters (HA-VE), which enabled fast 2PP-fabrication of 3D hydrogel constructs with μm-scale accuracy. A series of HA-VE macromers with tunable degrees of substitution were synthesized by lipase-catalyzed transesterification. HA-VE gels were proved to be injectable, photocurable, enzymatically degradable and mechanically comparable to various soft tissues. Owing to the unique molecular design, degradation products of HA-VE gels through hydrolysis are non-toxic polyvinyl alcohol and adipic acid. Furthermore, HA-VE gels were systematically characterized and compared to HA-acrylates (HA-AC) and HA-methacrylates (HA-MA) gels including macromer cytotoxicity, photoreactivity, swelling, and gel stiffness. Cytotoxicity assay with L929 fibroblasts revealed that HA-VE was significantly less toxic than HA-AC (P < 0.01) and HA-MA (P < 0.05). Crosslinking efficiency of HA-VE was comparable to HA-AC and much higher than HA-MA. Although the reactivity of HA-VE for homopolymerization was insufficient for 2PP, it was demonstrated that thiol–ene chemistry could substantially improve its reactivity. This optimization led to 2PP-fabrication of a HA-VE hydrogel construct with μm-scale accuracy. Low cytotoxicity, high reactivity and good biodegradability makes HA-VE promising candidates for biological applications in cell culture and tissue engineering.

Efficient Curing of Vinyl Carbonates by Thiol‐Ene Polymerization

Vinyl carbonates have recently been identified as a suitable alternative to (meth)acrylates, especially due to the low irritancy and cytotoxicity of these monomers. The drawback of some vinyl carbonates containing abstractable hydrogens arises through their moderate reactivity compared with acrylates. Within this paper, we use the thiol-ene concept to enhance the photoreactivity of vinyl carbonates to a large extent to reach the level of those of similar acrylates. Mechanical properties of the final thiol-ene polymers were determined by nanoindentation. Furthermore, low toxicity of all components was confirmed by osteoblast cell culture experiments.

Additive manufacturing of photosensitive hydrogels for tissue engineering applications

Abstract Hydrogels are extensively explored as scaffolding materials for 2D/3D cell culture and tissue engineering. Owing to the substantial complexity of tissues, it is increasingly important to develop 3D biomimetic hydrogels with user-defined architectures and controllable biological functions. To this end, one promising approach is to utilize photolithography-based additive manufacturing technologies (AMTs) in combination with photosensitive hydrogels. We here review recent advances in photolithography-based additive manufacturing of 3D hydrogels for tissue engineering applications. Given the importance of materials selection, we firstly give an overview of water-soluble photoinitiators for single- and two-photon polymerization, photopolymerizable hydrogel precursors and light-triggered chemistries for hydrogel formation. Through the text we discuss the design considerations of hydrogel precursors and synthetic approaches to polymerizable hydrogel precursors of synthetic and natural origins. Next, we shift to how photopolymerizable hydrogels could integrate with photolithography-based AMTs for creating well-defined hydrogel structures. We illustrate the working-principles of both single- and two-photon lithography and case studies of their applications in tissue engineering. In particular, two-photon lithography is highlighted as a powerful tool for 3D functionalization/construction of hydrogel constructs with μm-scale resolution. Within the text we also explain the chemical reactions involved in two-photon-induced biofunctionalization and polymerization. In the end, we summarize the limitations of available hydrogel systems and photolithography-based AMTs as well as a future outlook on potential optimizations.