Patterned biomaterial degradation to guide multicellular response, extracellular matrix deposition and in-vivo tissue formation.
Final Report Abstract
The cell microenvironment or cell niche includes the extracellular matrix (ECM), biochemical factors and neighboring cells, and the interaction between these three regulates cell function. Biomaterial scaffolds and hydrogels allow independent control of biochemical and biophysical properties and have contributed to the understanding of how cells sense these cues in tissue regeneration. To investigate how the biophysical properties of the ECM guide cell response and in-vivo tissue formation, we engineer the cell microenvironment using tunable alginate-based hydrogels. Degradability is of fundamental importance in regeneration, cell and drug delivery. Calcium crosslinked alginate hydrogels with a stiffness optimized for osteogenic differentiation were used for the regeneration of critical sized bone defects. While a trend was observed for hydrogel stiffness influencing bone formation, this effect was insufficient for complete healing. One possible limitation was that ionically crosslinked gels exhibited uncontrolled degradation via diffusion of calcium ions, and therefore the mechanical cues were not maintained in time. To overcome this limitation, we developed an alginate-based material system with two different types of covalent crosslinking and degradation modes. The first one involved oxidizing and adding norbornene or tetrazine functional groups to the polymer backbone. When combined, these species crosslinked spontaneously via Diels-Alder chemistry resulting in a hydrolytically-degradable hydrogel. The second one required the design of peptide crosslinkers that could be recognized and cleaved by native enzymes. When these crosslinkers were mixed with norbornene-modified alginate and exposed to UV light, an enzymatically-degradable hydrogel was formed by thiol-ene reaction. In both cases, mechanical and degradation properties could be tuned, and high viability of 3D encapsulated cells was confirmed. In-vivo tissue infiltration was shown to occur only in materials designed to degrade, not in non-degradable controls. The two orthogonal covalent crosslinking modes described above could then be combined. Taking advantage of the fact that one occurred spontaneously and the other relied on UV light for initiation, more sophisticated patterned biomaterials could be formed via photomasks. We generated spatial patterns of biophysical and biochemical properties, as confirmed by cell attachment, osteogenic or adipogenic differentiation, and patterned in-vivo tissue infiltration - an important step towards guided regeneration. Finally, and as proof-of-concept, we dual crosslinked these gels with covalent and ionic crosslinking, allowing for dynamic control of mechanical properties, with gels undergoing stiffening-softening cycles by adding and quenching calcium ions. Furthermore, these gels were functionalized with full-length fibronectin and protease-degradable sequences, offering applications in a variety of biomedical contexts. This novel and versatile biomaterial platform provides the basis for our current and future research on 3D in-vitro models of disease and regeneration and is currently continued in the following projects: DFG Emmy-Noether project: “Extracellular matrix biophysical cues in dormancy and bone metastasis”; DFG project “Directed cellular self-organization for advancing bone regeneration”, subproject “Hydrogels with spatial patterning of biophysical and biochemical properties to guide osteogenesis”.
Publications
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“In situ tissue regeneration through SDF-1α driven cell recruitment and stiffness-mediated bone regeneration in a critical-sized segmental femoral defect”, Acta Biomater, 2017, 60:50-63
A. Cipitria, K. Boettcher, S. Schönhals, D.S. Garske, K. Schmidt-Bleek, A. Ellinghaus, A. Dienelt, A. Peters, M. Mehta, C. Madl, N. Huebsch, D.J. Mooney, G.N. Duda
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“Mechanotransduction and growth factor signalling to engineer cellular microenvironments”, invited progress report, Adv Healthc Mater, 2017, 6(15):1700052
A. Cipitria, M. Salmeron-Sanchez
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“Hydrolytically-degradable click-crosslinked alginate hydrogels”, Biomaterials, 2018, 181:189-198
A. Lueckgen, D.S. Garske, A. Ellinghaus, R.M. Desai, A.G. Stafford, D.J. Mooney, G.N. Duda, A. Cipitria
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“Enzymatically-degradable alginate hydrogels promote cell spreading and in-vivo tissue infiltration”, Biomaterials, 2019, 217:119294
A. Lueckgen, D.S. Garske, A.E. Ellinghaus, D.J. Mooney, G.N. Duda, A. Cipitria
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“Alginate hydrogels for in-vivo bone regeneration: The immune competence of the animal model matters”, Tissue Engineering Part A, 2020, 26(15-16):852-862
D. Garske, K. Schmidt-Bleek, A. Ellinghaus, A. Dienelt, L. Gu, D.J. Mooney, G.N. Duda, A. Cipitria
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“Dual alginate crosslinking for local patterning of biophysical and biochemical properties”, Acta Biomater, 2020, 115:185-196
A. Lueckgen, D.S. Garske, A.E. Ellinghaus, D.J. Mooney, G.N. Duda, A. Cipitria
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“Dynamic Mechanical Control of Alginate-Fibronectin Hydrogels with Dual Crosslinking: Covalent and Ionic”, invitation to the special issue “Biodegradable Polymers for Medical Applications”, Polymers, 2021, 13(3), 433
S. Trujillo, M. Seow, A. Lueckgen, M. Salmeron-Sanchez, A. Cipitria
