The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G′ from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (≥40 vs ≥60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0–20% vs 75–100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extracellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field.

In the present work, gelatin type B is modified with highly reactive norbornene functionalities (Gel-NB) following a one-pot synthesis approach to enable subsequent thiol-ene photo-click crosslinking. The modification strategy displays close control over the amount of introduced functionalities. Additionally, Gel-NB exhibits considerably improved processing capabilities in terms of two-photon polymerization when benchmarked to earlier-reported crosslinkable gelatin derivatives (e.g., gelatin-methacrylamide (Gel-MOD) and gelatin-methacrylamide-aminoethylmethacrylate (Gel-MOD-AEMA)). The improvement is especially apparent in terms of minimally required laser power (20 mW vs ≥60 mW (Gel-MOD) vs ≥40 mW (Gel-MOD-AEMA) at 100 mm s scan speed) and processable concentration range (≥5 w/v% vs ≥10 w/v% (Gel-MOD/Gel-MOD-AEMA)). Furthermore, the proposed functionalization scheme maintains the excellent biocompatibility and cell interactivity of gelatin. Additionally, the norbornene functionalities have potential for straightforward postprocessing "thiol-ene" surface grafting of active molecules. As a consequence, a very promising material toward tissue engineering applications and more specifically, biofabrication, is presented.

Two‐photon polymerization (2PP) is a lithography‐based 3D printing method allowing the fabrication of 3D structures with sub‐micrometer resolution. This work focuses on the characterization of gelatin–norbornene (Gel–NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol‐ene system allows 2PP processing of cell‐containing materials at remarkably high scanning speeds (1000 mm s−1) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2–0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel–NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki‐67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high‐definition bioprinting of well‐defined constructs for long‐term cell culture studies.

The monoacylphosphineoxide (MAPO) salts Na-TPO and Li-TPO and the bisacylphosphineoxide (BAPO) salts BAPOONa and BAPO-OLi define an important and in the latter case a new class of water-soluble photoinitiators (PIs) for radical polymerization. These compounds showed excellent water-solubility of at least 29 g/L for Na-TPO and up to 60 g/L for BAPO-ONa in deionized water, thus exceeding the solubility of the state of the art PI for water-based systems Irgacure 2959 (I2959) 6-to 12-fold. However, biocompatibility, storage stability, and reactivity were equally important to replace the state of the art compounds. Concerning these properties, the MAPO and BAPO salts were at least in the same range (biocompatibility, stability) or showed even better results (reactivity) and had the additional advantage of visible light initiation. Na-TPO and Li-TPO achieved double bond conversions of an aqueous solution of N-acryloylmorpholine over 97% with broad band irradiation (320-500 nm), Li-TPO showed additionally very good biocompatibility (LC 50 5 3.1 mmol/L) and BAPO-OLi showed highest reactivity with visible light irradiation.

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A novel class of photopolymerizable hydrogel system based on hyaluronan vinyl esters has been developed for two-photon 3D microfabrication of biocompatible hydrogel constructs.

Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl (gel-MA) hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g. norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g. dithiothreitol). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerization which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. ⩾150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications.