Cell-Dense Bioink Design for Xolography: Coupling Refractive Index-Matching with Increased Photoreactivity

Bioxolography enables high-resolution fabrication of geometrically complex, cell-laden constructs for tissue engineering. However, tissue-relevant cell densities conflict with the optical transparency required for efficient dual-color volumetric printing. In this work, we extend the Bioxolography toolbox to include refractive index (RI) matching for cell-laden bioresins using iodixanol (IDX). Remarkably, IDX enhances optical transparency and boosts reactivity — a phenomenon unique to Xolography. Yet, excessive IDX compromises dual-color efficiency through increased absorption and undesired UV-only curing, underscoring a central trade-off between optical clarity and photochemical performance. Systematic tuning of resin compositions along an iso-refractive index line demonstrated the versatility of Bioxolography, with IDX enhancing polymerization and 4-Hydroxy-TEMPO providing biocompatible inhibition. Optimizing composition and printing parameters yielded GelMA hydrogels with cell densities up to 5·10 ⁶ cells·mL ⁻¹ . Cell-laden prints achieved sub-100 µm resolution and complex geometries such as channels and gyroids. Using skeletal muscle tissue as a model, we validated RI matched Bioxolography as a promising strategy for tissue engineering by demonstrating cell alignment along printed grooves and formation of mature muscle fibers characterized by MyoHC+ staining and fusion index. By integrating physical, chemical, and biological perspectives, this work advances Xolography toward biomaterials development and reinforces its position as an emerging volumetric (bio)printing technology.