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Acrylate-endcapped urethane-based precursors constituting a poly(D,L-lactide)/poly(ε-caprolactone) (PDLLA/PCL) random copolymer backbone are synthesized with linear and star-shaped architectures and various molar masses. It is shown that the glass transition and thus the actuation temperature could be tuned by varying the monomer content (0–8 wt% ε-caprolactone, T g,crosslinked = 10—42 °C) in the polymers. The resulting polymers are analyzed for their physico-chemical properties and viscoelastic behavior (G′ max = 9.6–750 kPa). The obtained polymers are subsequently crosslinked and their shape-memory properties are found to be excellent (R r = 88–100%, R f = 78–99.5%). Moreover, their potential toward processing via various additive manufacturing techniques (digital light processing, two-photon polymerization and direct powder extrusion) is evidenced with retention of their shape-memory effect. Additionally, all polymers are found to be biocompatible in direct contact in vitro cell assays using primary human foreskin fibroblasts (HFFs) through MTS assay (up to ≈100% metabolic activity relative to TCP) and live/dead staining (>70% viability).

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures

‘Organ-on-chip’ devices which integrate three-dimensional (3D) cell culture techniques with microfluidic approaches have the capacity to overcome the limitations of classical 2D platforms. Although several different strategies have been developed to improve the angiogenesis within hydrogels, one of the main challenges in tissue engineering remains the lack of vascularization in the fabricated 3D models. The present work focuses on the high-definition (HD) bioprinting of microvascular structures directly on-chip using two-photon polymerization (2PP). 2PP is a nonlinear process, where the near-infrared laser irradiation will only lead to the polymerization of a very small volume pixel (voxel), allowing the fabrication of channels in the microvascular range (10–30 µm in diameter). Additionally, 2PP not only enables the fabrication of sub-micrometer resolution scaffolds but also allows the direct embedding of cells within the produced structure. The accuracy of the 2PP printing parameters were optimized in order to achieve high-throughput and HD production of microfluidic vessel-on-chip platforms. The spherical aberrations stemming from the refractive index mismatch and the focusing depth inside the sample were simulated and the effect of the voxel compensation as well as different printing modes were demonstrated. Different layer spacings and their dependency on the applied laser power were compared both in terms of accuracy and required printing time resulting in a 10-fold decrease in structuring time while yielding well-defined channels of small diameters. Finally, the capacity of 2PP to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards. The functionality of the formed vessels was demonstrated with immunostaining of vascular endothelial cadherin (VE-Cadherin) endothelial adhesion molecules in both static and perfused culture.

Various biopolymers, including gelatin, have already been applied to serve a plethora of tissue engineering purposes. However, substantial concerns have arisen related to the safety and the reproducibility of these materials due to their animal origin and the risk associated with pathogen transmission as well as batch-to-batch variations. Therefore, researchers have been focusing their attention toward recombinant materials that can be produced in a laboratory with full reproducibility and can be designed according to specific needs (e.g., by introducing additional RGD sequences). In the present study, a recombinant protein based on collagen type I (RCPhC1) was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via two-photon polymerization (2PP). The results indicated a clear difference in 2PP processing capabilities between the chain-growth-polymerized RCPhC1-MA and the step-growth-polymerized RCPhC1-NB/SH. More specifically, reduced swelling-related deformations resulting in a superior CAD-CAM mimicry were obtained for the RCPhC1-NB/SH hydrogels. In addition, RCPhC1-NB/SH allowed the processing of the material in the presence of adipose tissue-derived stem cells that survived the encapsulation process and also were able to proliferate when embedded in the printed structures. As a consequence, it is the first time that successful HD bioprinting with cell encapsulation is reported for recombinant hydrogel bioinks. Therefore, these results can be a stepping stone toward various tissue engineering applications.

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl 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.. DTT). 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 in which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerisation 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. Creative Commons Attribution license.

Direct cell encapsulation is a powerful tool for fabrication of biomimetic 3D cell culture models in vitro. This method allows more precise recapitulation of the natural environment and physiological functions of cells compared to classical 2D cultures. In contrast to seeding cells on prefabricated scaffolds, cell encapsulation offers benefits regarding high initial cell loading, uniformity of cell distribution and more defined cell–matrix contact. Two-photon polymerization (2PP) based 3D printing enables the precise engineering of cell-containing hydrogel constructs as tissue models. Two-photon initiators (2PIs) specifically developed for this purpose still exhibit considerable cyto- and phototoxicity, impairing the viability of encapsulated cells. This work reports the development of the first cleavable diazosulfonate 2PI DAS, largely overcoming these limitations. The material was characterized by standard spectroscopic methods, white light continuum two-photon absorption cross-section measurements, and its photosensitization of cytotoxic singlet oxygen was compared to the well-established 2PI P2CK. When DAS is used at double concentration to compensate for the lower two-photon cross section, its performance in 2PP-printing of hydrogels is similar to P2CK based on structuring threshold and structure swelling measurements. PrestoBlue metabolic assay showed vastly improved cytocompatibility of DAS in 2D. Cell survival in 3D direct encapsulation via 2PP was up to five times higher versus P2CK, further demonstrating the excellent biocompatibility of DAS and its potential as superior material for laser-based biofabrication.

The z-scan technique has become a standard method to measure 2-photon absorption (2PA) properties of materials used for 2-photon applications. Here we present a completely automated, easily tunable z;-scan setup for spectral scanning. An algorithm collecting the required laser beam parameters allows to reliably determine the optimal working window of newly synthesized 2PA photoinitiators (PI) used for two-photon polymerization (2PP) in a wide spectral range. A complete spectrum (3 measurements per wavelength in 10 nm steps) can be obtained within an hour. Matching the wavelength used for 2PP to the maximum 2PA significantly increased the 2PP performance of the system. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License.

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

Photodynamic therapy (PDT) involves a photosensitizing agent activated with light to induce cell death. Two-photon excited PDT (TPE-PDT) offers numerous benefits compared to traditional one-photon induced PDT, including an increased penetration depth and precision. However, the in vitro profiling and comparison of two-photon photosensitizers (PS) are still troublesome. Herein, we report the development of an in vitro screening platform of TPE-PS using a 3D osteosarcoma cell culture. The platform was tested using three different two-photon (2P) active compounds - a 2P sensitizer P2CK, a fluorescent dye Eosin Y, and a porphyrin derivative (TPP). Their 2P absorption cross-sections (σ2PA) were characterised using a fully automated z-scan setup. TPP exhibited a remarkably high σ2PA at 720 nm (8865 GM) and P2CK presented a high absorption at 850 nm (405 GM), while Eosin Y had the lowest 2P absorption at the studied wavelengths (

Over the recent decades gelatin has proven to be very suitable as an extracellular matrix mimic for biofabrication and tissue engineering applications. However, gelatin is prone to dissolution at typical cell culture conditions and is therefore often chemically modified to introduce (photo-)crosslinkable functionalities. These modifications allow to tune the material properties of gelatin, making it suitable for a wide range of biofabrication techniques both as a bioink and as a biomaterial ink (component). The present review provides a non-exhaustive overview of the different reported gelatin modification strategies to yield crosslinkable materials that can be used to form hydrogels suitable for biofabrication applications. The different crosslinking chemistries are discussed and classified according to their mechanism including chain-growth and step-growth polymerization. The step-growth polymerization mechanisms are further classified based on the specific chemistry including different (photo-)click chemistries and reversible systems. The benefits and drawbacks of each chemistry are also briefly discussed. Furthermore, focus is placed on different biofabrication strategies using either inkjet, deposition or light-based additive manufacturing techniques, and the applications of the obtained 3D constructs. STATEMENT OF SIGNIFICANCE: Gelatin and more specifically gelatin-methacryloyl has emerged to become one of the gold standard materials as an extracellular matrix mimic in the field of biofabrication. However, also other modification strategies have been elaborated to take advantage of a plethora of crosslinking chemistries. Therefore, a review paper focusing on the different modification strategies and processing of gelatin is presented. Particular attention is paid to the underlying chemistry along with the benefits and drawbacks of each type of crosslinking chemistry. The different strategies were classified based on their basic crosslinking mechanism including chain- or step-growth polymerization. Within the step-growth classification, a further distinction is made between click chemistries as well as other strategies. The influence of these modifications on the physical gelation and processing conditions including mechanical properties is presented. Additionally, substantial attention is put to the applied photoinitiators and the different biofabrication technologies including inkjet, deposition or light-based technologies.

The in situ observation of cell movements and morphological parameters over longer periods of time under physiological conditions is critical in basic cell research and biomedical applications. The quantitative phase-contrast microscope applied in this study has a remarkably small size, therefore it can be placed directly into a humidified incubator. Here, we report on the successful application of this M4 Holomonitor to observe cancer cell motility, motility speed, and migration in the presence of the green tea polyphenol, epigallocatechin gallate, as well as to monitor the adhesion of preosteoblast cells on nanostructured titanate coatings, relevant for biomedical applications. A special mechanical stage was developed to position the sample into that range of the optical arrangement where digital autofocusing works with high reproducibility and precision. By in-depth analyzing the obtained single cell morphological parameters, we show that the limited vertical resolution of the optical setup results in underestimated single cell contact area and volume and overestimated single cell averaged thickness. We propose a simple model to correct the recorded data to obtain more precise single cell parameters. We compare the results with the kinetic data recorded by a surface sensitive optical biosensor, optical waveguide lightmode spectroscopy.

Arylhydrazines react with excess nitrous acid to form mixtures of the diazonium ion and aryl azide. By use of the weakly basic 2,4-dinitrophenylhydrazine (DNP), pKa= 1.55, information about the mechanism of the diazotisation reaction has been obtained. Three successive stages can be observed. The initial reaction consists of parallel nitrosations by N2O3 and NO+ of the free base DNP at the terminal nitrogen. This is followed by a stage with the rate independent of [HNO2], almost certainly a tautomerisation, probably to form ArNNNHOH and ArNHNNOH. The former is converted to ArN3 while the latter undergoes a further electrophilic nitrosation by NO+ of a conjugate base species to yield a di-N,N′-nitrosoarylhydrazine, a precursor to the diazonium ion.