Engineering honeycomb-like scaffolds via direct laser writing for cardiac tissue engineering
Embargo Date
2026-06-26
OA Version
Citation
Abstract
Cardiac tissue engineering has emerged with the goal of replacing damaged areas in the heart with engineered functional cardiac patches. Cardiac muscle has a complex 3D tissue structure composed of various cells including cardiomyocytes, fibroblasts and endothelial cells that are anchored to a highly aligned extracellular matrix (ECM). Several approaches in cardiac tissue engineering involve the fabricating a scaffold to guide cell alignment. However, replicating the aligned fibrillar ECM network at the microscale, typically ranging from 10 to 100 μm, remains a significant challenge. Current limitations can be attributed to the poor spatial resolution of the reported fabrication techniques and have motivated tissue engineers to explore new technologies for fabricating cardiac tissue structures. Inspired by emerging trends in cardiac tissue engineering, we applied a commercial two- photon direct laser writing (DLW) technique to fabricate high-resolution structures for cellular microenvironments. We made suspended scaffold platforms with different unit cells, and generated cardiac tissues upon them using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Next, our investigation examined the influence of unit cell geometry on sarcomere alignment. To conduct this analysis, we employed Sarc-Graph, a Python package tailored for automated quantitative assessment of fluorescently labeled Z- discs, in hiPSC-CMs. Previous studies have suggested that the fibrous network of proteins within cardiomyocytes exhibits an aligned architecture, resembling the microstructural organization found in heart tissue. Therefore, we hypothesized that early-stage iPSC-CMs seeded within honeycomb scaffolds with unit pores close to the size of mature cardiomyocytes will organize and align, ultimately resulting in functional cardiac tissue scaffolds. We have found that sarcomeres in iPSC-CMs align more efficiently within elongated unit pores of scaffolds compared to hexagonal ones, as well as compared to cells that are not interacting with any scaffolds. Also, beating cardiomyocytes bend the scaffold, suggesting that the scaffolds are strong enough to support the cells while remaining flexible to accommodate their movement as they beat. Additionally, introducing micron-scale windows in the scaffold walls enables cell-cell interactions across multiple unit cells. Overall, this research provides a simplified solution for fabricating porous, high-resolution 3D scaffolds with controllable mechanical properties for cardiac tissue engineering using DLW technology.