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.

三个双正交点击反应、光引发硫醇-炔反应、叠氮-炔环加成反应和甲基四嗪-反式环辛烯Diels Alder反应，用于独立控制水凝胶支架中几种生物活性蛋白向瓣膜间质细胞（VIC）的呈递。研究发现，束缚成纤维细胞生长因子 (FGF-2) 在浓度为 10 ng/mL 时可抑制肌成纤维细胞活化（从 48 ± 7% 至 17 ± 6%）并促进增殖（从 10 ± 2% 至 54 ± 3%） 。在促纤维化细胞因子转化生长因子-β (TGF-β1) 存在的情况下，FGF-2 可以保护 VIC 成纤维细胞表型，即使 TGF-β1 浓度远高于 FGF-2。对于纤维钙化 VIC 表型，发现 TGF-β1 和骨形态发生蛋白 2 (BMP-2) 可以协同促进钙化结节形成（与单独使用 TGF-β1 或 BMP-2 相比，结节增加五倍） 。利用正交点击反应，FGF-2、TGF-β1 和 BMP-2 组合被图案化到水凝胶上的不同区域，以控制 VIC 激活和结节形成。同一支架的不同区域之间的细胞串扰受到每个区域的大小以及不同区域之间的界面面积的影响。总的来说，这些结果证明了光引发硫醇炔反应对模板侧链功能的多功能性和稳健性，允许多种蛋白质的生物共轭。这种方法保持了蛋白质的生物活性，提供了一个体外平台，能够更好地理解组织纤维化所涉及的复杂机制。