Kirsten Borchers1 2 Annika Wenz3 Eva Hoch3 Christiane Claassen2 Lisa Sewald2 Achim Weber1 2

1, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, , Germany
2, University of Stuttgart, Stuttgart, , Germany
3, University of Stuttgart (former address), Stuttgart, , Germany

Digital 3D manufacturing techniques are being successfully adapted for tissue engineering applications. Each technique requires printable matrices, so called bioinks that match with the requirements of the process and meet the needs of cells. We provide bioinks based on biomolecules from the native extracellular matrix and present a well-controlled procedure to produce gelatin derivatives with defined degrees of modification.[1]
Double chemical functionalization of gelatin by methacryloylation and acetylation enabled control over the viscous behavior of gelatin solutions and the photochemical crosslinking. [2] For the first time we can quantify the total amounts of coupled methacrylate functions (methacrylic acid coupled to hydroxyl groups of the biomolecule), methacrylamide functions (methacrylic acid coupled to amino groups) and acetic functions based on 2D NMR.[1] E.g. application of 10-fold molar excess of methacrylic anhydride in relation to free amino groups resulted in GM10, gelatin derivatives with high degrees of metharcyloylation (DM) of 0.958 ± 0.068 mmol methacryl-functions per gram gelatin. Application of 5-fold or 2-fold excess lead to GM5 or GM2 with 0.338 ± 0.022 mmol/g or 0.618 ± 0.032 mmol/g, respectively.
It is of note that controlled addition of acetic anhydride yielded GM2A8 or GM5A5 with the same DM as GM2 or GM5, respectively, while the total degrees of modification (DMA) were equal to the DMA of GM10.
Viscosity analysis of gelatin solutions showed that the viscosity decreased with increasing DMA from 6.17 ± 2.20 mPas (GM2) to 2.42 ± 0.205 mPas (GM2A8) for 10 % solutions and even stronger from 110.2 ± 31.5 mPas (GM2) to 9.0 ± 0.4 mPas (GM2A8) for 20 % solutions.[2] The upper viscosity limit reported for inkjet printing is approx. 10 mPas. We reached the ink-jet printable range by double functionalization, which was not possible with simple functionalization at comparable DM. Through specification of the degrees of methacryloylation and acetylation we can provide bioinks with various viscosities, gelling behavior and crosslinking potential for different printing methods. Inkjet printing of low viscous GM10 inks loaded with chondrocytes indicated that the presented bioink is generally applicable for inkjet processing of viable cells.[2] Stable hydrogel crosslinking was achieved with layerwise extrusion printing and irradiation of bioinks composed of GM5A5 and GM2A8, photoinitiator and gradients of methacrylated chondroitin sulfate and hyaluronic acid. Promotion of osteogenic differentiation of mesenchymal stem cells was achieved in hydroxyapatite containing formulations of GM2, GM5 and methacrylated hyaluronic acid.[3]

1 Claassen et al., Biomacromolecules, 2017 under revision.
2 Hoch et al., J Mater Chem B, 2013. 1(41) 5675-5685.
3 Wenz et al., Biofabrication, 2017 accepted.