Cell-instructive hydrogel gradient arrays
Final Report Abstract
Spatio-temporally varying mechanical and biomolecular cues of the extracellular microenvironment determine cell fate decisions in living tissues and thus need to be recapitulated in effectively cellinstructive materials. Towards this aim, we executed an interdisciplinary research program to employ for the first time a set of in situ forming, growth factor-affine starPEG-GAG hydrogels in combination with advanced 3D printing and microfluidics methodology for (i) producing bioresponsive polymer matrices with orthogonally graded mechanical and biomolecular characteristics, (ii) integrating graded GAG-hydrogel into 3D cell culture platforms, and (iii) performing parallelized studies of matrix-controlled cellular response patterns. The fundamental principles of molecular transport and binding in the GAG-based hydrogels were unraveled in the first part of the project and applied to form and characterize cell-instructive biomolecular gradients within the matrices. To predict and evaluate spatio-temporal morphogen distributions in multiphasic systems, we developed and applied reaction-diffusion models in COMSOL Multiphysics. These models were parametrized with diffusion coefficients and binding constants by Fluorescence Recovery After Photobleaching, Microscale Thermophoresis, and Bio-Layer-Interferometry. Biophysical and biomolecular gradients were implemented in cell-instructive GAG-hydrogels using casting, additive manufacturing, and microfluidic techniques whereas special emphasis was on the development of graded signals of soluble signalling molecules (growth factors and cytokines). Graded materials were applied to control embedded cell morphology placing emphasis on the morphogenesis of human umbilical vein endothelial cells into capillary networks. In addition to the original plan, we developed a printable ternary hydrogel system with tunable overall charge density and variable charge density at the GAG building block as wells as in situ crosslinkable binary hydrogels with variable sulfation pattern since it turned out that fine tuning of the hydrogel sulfation pattern could be used as most powerful tool to modulate and even program cytokine gradients and factor sequestration within GAG-hydrogel matrices. Moreover, we could show that such GAG-based hydrogels efficiently protect and preserve embedded signalling molecules against proteolytic degradation. Furthermore, we developed an inkjet-based method for 3D printing of multicomponent GAG-hydrogels that allows to grade the biophysical and biomolecular composition in zonal scaffolds with unprecedented (~50 µm) resolution. The materials pave the way for more realistic cell culture and organoid models. As a first step toward this objective, we performed 3D cell culture experiments to analyse the influence of graded GAG-hydrogel signals on the migration and morphogenesis of human mesenchymal stems cells, the polarization of mammary epithelial cells, and the promotion of renal tubulogenesis. As such, the results of the in here summarized project will be the base for both PI’s future research projects to tailor cell-instructive GAG based hydrogel materials for advanced in vitro models and the development of biomedical applications for regenerative and personalized medicine. Key findings are (1) the developed toolbox to produce hydrogel arrays that will allow for further studies to characterize the interplay of orthogonally matrix signals, (2) the established models of molecular transport that also should help to tailor GAG-matrices for in vivo applications, (3) gradually adjusted binary and ternary hydrogel systems that should allow to translate the findings of orthogonally adjusted matrix signals into powerful in situ polymerizable and therefore for in vivo applications well suited matrices that may pave the way for new concepts for cell-instructive materials that can program orthogonally matrix signals.
Publications
- (2019) High resolution bioprinting of multi-component hydrogels. Biofabrication 11 (4) 045008
Zimmermann, Ralf; Hentschel, Christoph; Schrön, Felix; Moedder, Denise; Büttner, Teresa; Atallah, Passant; Wegener, Thomas; Gehring, Thomas; Howitz, Steffen; Freudenberg, Uwe; Werner, Carsten
(See online at https://doi.org/10.1088/1758-5090/ab2aa1) - StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4. Advanced Healthcare Materials 2016, 40, 8861–8891
L. Schirmer, P. Atallah, C. Werner, U. Freudenberg
(See online at https://doi.org/10.1002/adma.201601908) - Heparin-based hydrogels induce human renal tubulogenesis in vitro. Acta Biomaterialia 2017, 57, 59–69
H. Weber, M. Tsurkan, V. Magno, U. Freudenberg, C. Werner
(See online at https://doi.org/10.1016/j.actbio.2017.05.035) - Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro. Biomaterials 2017, 112, 20–30
M. Nowak, U. Freudenberg, M. Tsurkan, C. Werner, K. Levental
(See online at https://doi.org/10.1016/j.biomaterials.2016.10.007) - Exploring Structure–Property Relationships of GAGs to Tailor ECM-Mimicking Hydrogels. Polymers 2018, 10, 1376
R. Zimmermann, C. Werner, J. Sterling
(See online at https://doi.org/10.3390/polym10121376) - In situ-forming, cell-instructive hydrogels based on glycosaminoglycans with varied sulfation patterns. Biomaterials 2018, 181, 227–239
P. Atallah, L. Schirmer, M. Tsurkan, Y.D.P. Limasale, R. Zimmermann, C. Werner, U. Freudenberg
(See online at https://doi.org/10.1016/j.biomaterials.2018.07.056) - Charge-tuning of glycosaminoglycan-based hydrogels to program cytokine sequestration. Faraday Discuss. 2019
U. Freudenberg, P. Atallaha, Y. D. P. Limasale, C. Werner
(See online at https://doi.org/10.1039/c9fd00016j)
