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

Abstract

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{middle dot}106 cells{middle dot}mL-1. Cell-laden prints achieved sub-100 {micro}m 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. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/729865v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@19c0380org.highwire.dtl.DTLVardef@1a9e920org.highwire.dtl.DTLVardef@84210org.highwire.dtl.DTLVardef@294112_HPS_FORMAT_FIGEXP M_FIG C_FIG For printing higher cell density bioresins with Xolography, iodixanol (IDX) is added for refractive index-matching. The addition leads to an unexpected additional effect with increased reactivity in the dual-color photopolymerization. With careful adjustment of the resin composition and the printing parameters, Bioxolography is proven as a viable tool for tissue engineering.