HRP-crosslinked silk-gelatin bioinks: printability dynamics and modulation of stem cell lineage commitment in 3D...

Advancements in 3D bioprinting demand bioinks that demonstrate not only precise printability and mechanical robustness but also consistent biochemical and cellular performance. From previous iterations in our laboratory, a silk fibroin-gelatin (SF-G) bioink was conjugated with mushroom tyrosinase (MT) as the enzymatic crosslinker. However, its clinical translation was hindered by several limitations, including batch-to-batch variation in concentration and enzymatic activity, protracted gelation kinetics, and inconsistent rheological and structural characteristics. To overcome these challenges, we developed a next-generation SF-G bioink employing horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂) as an alternative enzymatic crosslinking system. This approach facilitated rapid and tunable crosslinking through β-sheet enhancement, yielding hydrogels with improved stiffness, shape fidelity, and resistance to enzymatic degradation. Detailed rheological analysis confirmed optimal shear-thinning behaviour and print fidelity, while reactive oxygen species (ROS) quantification ensured cytocompatibility across physiologically relevant concentrations. Human bone marrow-derived mesenchymal stem cells (hBMSCs) encapsulated within the constructs maintained high viability and demonstrated robust osteogenic and chondrogenic lineage commitment. Furthermore, supplementation with triiodothyronine (T3) and transforming growth factor-β3 (TGF-β3) augmented matrix deposition and tissue-specific morphogenesis. Notably, a 10 U HRP-H₂O₂ formulation emerged as the most promising candidate, offering a strategic balance of mechanical integrity, bioactivity, and reproducibility. This optimized enzymatic system paves the way for scalable and clinically viable SF-G bioinks tailored for advanced tissue engineering and regenerative medicine applications.