Biomaterial degradation is a key biophysical cue which influences stem cell behavior and tissue formation in-vitro. However, it is still unclear what degradation properties optimize multicellular response, extracellular matrix (ECM) deposition and in-vivo tissue formation. Our goal is to gain spatial and temporal control of the mechanism and kinetics of hydrogel degradation to direct cell infiltration and guide ECM formation.
Covalently click-crosslinked alginate hydrogels were fabricated with norbornene (N) and tetrazine (T) reacting functional groups . Alginate chains were oxidized to impart passive hydrolytic degradation and the degradation rate could be adjusted by the percent of oxidation. The materials were characterized for gelation kinetics, rheological properties, elastic modulus measured by unconfined compression testing and swelling behavior. The degree of crosslinking and resulting mechanical properties were controlled by: (i) polymer concentration (2 wt/v %), (ii) the amount of norbornene, as determined by nuclear magnetic resonance spectroscopy (low vs. high) and (iii) the stoichiometry ratio of norbornene and tetrazine (N:T = 0.5 - 3).
The materials were further functionalized by adding Arg-Gly-Asp (RGD) sequences that allow for cell attachment. These sites were incorporated in two distinct ways: (i) attachment to the polymer backbone via carbodiimide chemistry during synthesis or (ii) linkage to the norbornene group via thiol-ene chemistry during UV treatment. MC3T3-E1 mouse pre-osteoblasts were seeded on 2D and encapsulated in 3D, non-degradable, RGD-modified alginates via carbodiimide chemistry (peptides / polymer chain = 20). Cell viability and morphology was monitored at days 1 and 7. Cell spreading was observed on 2D, while 3D encapsulated cells remained round and viable, as expected for non-degradable, elastic hydrogels. Furthermore, cells were seeded on 2D gels, incubated with 2mM cysteine-bearing RGD peptides and exposed to UV light. The surface modification was confirmed by the cell attachment and spread morphology after day 3. Ongoing in-vitro assays include cell proliferation, invasion assay into the 3D matrix and surface patterning of RGD peptide via UV and photomasks.