Bioorthogonal click chemistries enable simultaneous spatial patterning of multiple proteins to probe synergistic protein effects on fibroblast function.

Three biorthogonal click reactions, a photoinitiated thiol-yne reaction, an azide-alkyne cycloaddition, and a methyltetrazine-transcyclooctene Diels Alder, were used to independently control the presentation of several bioactive proteins to valvular interstitial cells (VICs) in hydrogel scaffolds. Tethered fibroblast growth factor (FGF-2) was found to suppress myofibroblast activation (from 48 ± 7% to 17 ± 6%) and promote proliferation (from 10 ± 2% to 54 ± 3%) at a concentration of 10 ng/mL. In the presence of the pro-fibrotic cytokine transforming growth factor-beta (TGF-β1), FGF-2 could protect the VIC fibroblast phenotype, even at much higher concentrations of TGF-β1 than that of FGF-2. With respect to the fibrocalcific VIC phenotype, TGF-β1 and bone-morphogenic protein-2 (BMP-2) were found to synergistically promote calcific nodule formation (a five-fold increase in nodules compared to TGF-β1 or BMP-2 alone). Exploiting the orthogonal click reactions, FGF-2, TGF-β1 and BMP-2 combinations were patterned into distinct regions on a hydrogel to control VIC activation and nodule formation. Cellular crosstalk between separate regions of the same scaffold was affected by the size of each region as well as the interfacial area between different regions. Collectively, these results demonstrate the versatility and robustness of a photoinitiated thiol-yne reaction to template pendant functionalities that allow for the bioconjugation of multiple proteins. This approach maintains protein bioactivity, providing an in vitro platform capable of achieving a better understanding of the complex mechanisms involved in tissue fibrosis.