Millimeter‐Scale Dual‐Opposing RNA‐Gradient Hydrogel for Interfacial Gene Silencing

ABSTRACT Tissue interface restoration poses significant challenges in tissue engineering, particularly in areas requiring gradients of cellularity, biochemical composition, and mechanical properties essential for tissue‐specific functions. Recent advancements in microfluidic technology have enabled the creation of hydrogels with spatially defined gradients of biological molecules for engineering gradient tissues that mimic the natural heterogeneity of the native extracellular matrix. However, these gradients are typically outside relevant millimeter length scales for biological interfaces. To address these challenges, a branched microfluidic system capable of generating millimeter‐scale hydrogels with dual‐opposing gradients of RNAi molecules nanocomplexed with thiolated polyethyleneimine is demonstrated. These nanocomplexes are incorporated into photocrosslinkable poly(ethylene glycol)‐diacrylate (PEG‐DA) monomer solutions, which are then injected and mixed within a PDMS microfluidic chip featuring a branched channel network. The process results in stable, linear, 3‐mm dual‐opposing RNA gradients within the hydrogel, which is subsequently photocrosslinked. The generated hydrogels demonstrate precise spatial regulation of gene expression within encapsulated cells, as confirmed by fluorescence analysis. This platform holds significant potential for engineering complex tissue constructs and enabling the targeted delivery of RNAi molecules, influencing both encapsulated and endogenous cells. This advancement could play a crucial role in the regeneration of critical tissue interfaces such as tendon‐to‐bone and cartilage‐to‐bone.