This work explores the utility of bioorthogonal tetrazine (Tz)/trans-cyclooctene (TCO) ligation to induce in-situ stiffening in a hydrogel construct to promote chondrogenic redifferentiation of encapsulated passaged chondrocytes. Passaged chondrocytes were encapsulated in hydrogels fabricated using Tz-functionalized hyaluronic acid (HA-Tz), norbornene-functionalized protease degradable peptide crosslinker (SMR-bisNb) and TCO-functionalized fibronectin derived peptide (RGD-TCO). The cells showed high viability across a 42-day culture. IVIS imaging was used to track cell-mediated hydrogel degradation by using fluorescent TCO-tags attached to the gel. No significant gel degradation was seen across the culture period, indicating high suitability of this gel platform for long term culture of these chondrocytes. To investigate the effect of matrix stiffening on cellular behavior and phenotype, the gel was stiffened in-situ at Day 7 during a 21-day culture using TCO-functionalized hyaluronic acid (HA-TCO). HA-TCO mediated stiffening of hydrogel resulted in a nearly two-fold increase in storage modulus of the hydrogel. This stiffening promoted an increase in cell circularity, reminiscent of native chondrocytes in articular cartilage (AC). Moreover, gel stiffening upregulated chondrogenic gene expression (COL2, ACAN, SOX9) while downregulating fibroblastic gene expression (COL1, ACTA2 and TNC). The stiff gel formulation also promoted the upregulation of deep AC zone marker COLX. Superficial AC zone marker PRG4 was upregulated in all 3D cultures as compared to 2D controls. On-going work is focused on utilizing the fast kinetics of Tz-TCO ligation to form a bilayered hydrogel construct with soft and stiff layers mimicking the stiffness gradient observed in native AC. The bilayered hydrogel will be used to assess if chondrocytes can be specifically tuned to express regional markers PRG4 and COLX respectively and promoting zone-specific extracellular matrix production to better mimic native AC.

Justin Parreno

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