Engineering three-dimensional (3D) hydrogels with well-defined architectures has become increasingly important for tissue engineering and basic research in biomaterials science. To fabricate 3D hydrogels with (sub)cellular-scale features, two-photon polymerization (2PP) shows great promise although the technique is limited by the selection of appropriate hydrogel precursors [1]. To mimic the extracellular matrix gelatin has been modified with low toxic vinylester groups [2]. 2PP of this hydrogel precursor has been confirmed in the presence of living tissue without harming them.[3] Essential for 2PP of hydrogels are suitable 2 photon initiators that are water-soluble [4,6]. To avoid the uptake of these initiators into the cell which can lead to serious damage of the cells, these photoinitiators where covalently bound to hyaluronan. [5] Very recently, hydrogels based on polymerizable hyaluronic acid vinylester [7] or on synthetic biocompatible polyvinyl alcohol [8] has been established. Besides the crosslinking of the hydrogel precursors in the presence of cells, the controlled degradation of hydrogels is a possiability to guide cells in the 3D construct [9]. Using the new 2 Photon Grafting technique it is possible to attach cell selective peptides in the 3D volume. [10-12]

[1] A. Ovsianikov, V. Mironov, J. Stampfl, R. Liska: "Engineering 3D cell-culture matrices: multiphoton processing technologies for biological and tissue engineering applications"; Expert Review of Medical Devices (invited), 9 (2012), 6; S. 613 - 633.

[2] X.H. Qin, J. Torgersen, R. Saf, S. Mühleder, N.U Pucher, S. Ligon, W. Holnthoner, H. Redl, A. Ovsianikov, J. Stampfl, R. Liska: "Three-Dimensional Microfabrication of Protein Hydrogels via Two-Photon-Excited Thiol-Vinyl Ester Photopolymerization"; Journal of Polymer Science Part A: Polymer Chemistry, 51 (2013), S. 4799 - 4810.

[3a] J. Torgersen, X.H. Qin, Z. Li, A. Ovsianikov, R. Liska, J. Stampfl: "Hydrogels for Two-Photon Polymerization: A Toolbox for Mimicking the Extracellular Matrix"; Advanced Functional Materials, 10 (2013), 6.

[3b] J. Torgersen, A. Ovsianikov, V. Mironov, N.U Pucher, X.H. Qin, Z. Li, K. Cicha, T. Machacek, V. Jantsch-Plunger, R. Liska, J. Stampfl: "Photo-sensitive hydrogels for threedimensional laser microfabrication in the presence of whole organisms"; Journal of Biomedical Optics, 17 (2012), 10; S. 1 - 10.

[4] Z. Li, J. Torgersen, A. Ajami, S. Mühleder, X.H. Qin, W. Husinsky, W. Holnthoner, A. Ovsianikov, J. Stampfl, R. Liska: "Initiation efficiency and cytotoxicity of novel water-soluble two-photon photoinitiators for direct 3D microfabrication of hydrogels"; RSC Advances, 36 (2013), S. 15939 - 15946.

[5] M. Tromayer, P. Gruber, M. Markovic, A. Rosspeintner, E. Vauthey, H. Redl, A. Ovsianikov, R. Liska: "A biocompatible macromolecular two-​photon initiator based on hyaluronan"; Polymer Chemistry, 8 (2017), 2; S. 451 - 460.

[6] M. Tromayer, A. Dobos, P. Gruber, A. Ajami, R. Dedic, A. Ovsianikov, R. Liska: "A biocompatible diazosulfonate initiator for direct encapsulation of human stem cells via two-photon polymerization"; Polymer Chemistry, 9 (2018), 22; S. 3108 - 3117.

[7] E. Zerobin, M. Markovic, Z. Tomasikova, X.H. Qin, D. Ret, P. Steinbauer, J. Kitzmüller, W. Steiger, P. Gruber, A. Ovsianikov, R. Liska, S. Baudis: "Hyaluronic acid vinyl esters: A toolbox toward controlling mechanical properties of hydrogels for 3D microfabrication"; Journal of Polymer Science Part A: Polymer Chemistry, 58 (2020), 8; S. 1288 - 1298.

[8] S. Baudis, D. Bomze, M. Markovic, P. Gruber, A. Ovsianikov, R. Liska: "Modular Material System for the Microfabrication of Biocompatible Hydrogels Based on Thiol-Ene-Modified Poly(vinyl alcohol)"; Journal of Polymer Science Part A: Polymer Chemistry, 54 (2016), 13; S. 2060 - 2070.

[9] M. Lunzer, L. Shi, O.G. Andriotis, P. Gruber, M. Markovic, P.J. Thurner, D. Ossipov, R. Liska, A. Ovsianikov: "A Modular Approach to Sensitized Two-Photon Patterning of Photodegradable Hydrogels"; Angewandte Chemie International Edition, 57 (2018), 46; S. 15122 - 15127.

[10] A. Ovsianikov, Z. Li, J. Torgersen, J. Stampfl, R. Liska: "Selective Functionalization of 3D Matrices Via Multiphoton Grafting and Subsequent Click Chemsitry"; Advanced Functional Materials, 22 (2012), 16; S. 3429 - 3433.

[11] A. Ovsianikov, Z. Li, A. Ajami, J. Torgersen, W. Husinsky, J. Stampfl, R. Liska: "3D grafting via three-photon induced photolysis of aromatic azides"; Applied Physics A: Materials Science & Processing, 108 (2012), 1; S. 29 - 34.

[12] Z. Li, E. Stankevičius, A. Ajami, G. Račiukaitis, W. Husinsky, A. Ovsianikov, J. Stampfl, R. Liska: "3D alkyne-azide cycloaddition: spatiotemporally controlled by combination of aryl azide photochemistry and two-photon grafting"; Chemical Communications, 49 (2013), S. 7635 - 7637.