• 3d-printer
• bioscaffolds
• chromatin
• histone modifications
• polycaprolactone
• STORM
• super-resolution microscopy

The aim of this study is to develop an imaging and analysis protocol to highlight the effects on chromatin organization induced by the interaction between cellular systems and 3D-printed scaffolds.
Chromatin, the condensed form of DNA around histones within the cell nucleus, can be examined at the nanoscale utilizing super-resolution imaging techniques. Recent studies have revealed that the substrate stiffness, namely the physical properties of the material surfaces where cells adhere and grow, can modulate chromatin organization. While the cellular environment is generally found to significantly affect the macroscopic cell function, the specific mechanisms through which biophysical cues regulate spatial nano-scale organization remain unclear.
Chromatin within the cell nucleus is visualized through Stochastic Optical Reconstruction Microscopy (STORM), a super-resolution technique that improves spatial resolution beyond the diffraction limit by localizing individual molecules. Based on the spatial coordinates of the molecules provided by STORM, two quantitative analyses are performed in this work: Cluster Analysis and Edge Analysis. These methods quantify how chromatin organizes itself into clusters and at nucleus boundaries, respectively. To test the first part of the implemented protocol, mouse embryonic stem cells (mESCs) are differentiated into neuronal progenitor cells (mNPCs), in which differences rise in terms of the organization of histone H3 modifications compared to mESCs. For the second part of the protocol, a 3D-Bioplotter is used to fabricate 3D-printed scaffolds made of polycaprolactone (PCL), a biocompatible polymeric material that can influence cell behavior. To achieve optimal printing as well as knowledge on the physical properties of the used polymer system, the printing rheology of PCL is modeled and studied to assess how the filament diameter depends on various printing factors. Then, the printing parameters are optimized and tuned to enhance polymer filament printability and shape fidelity during scaffold fabrication. Scaffolds composed of a single uniform layer, as well as scaffolds with precise 3D geometries are obtained and inspected by Scanning Electron Microscopy (SEM) and Profilometry. Finally, strategies are developed to visualize the nanoscale organization of chromatin in Human Intestinal Smooth Muscle Cells (HISMC) on 2D and 3D scaffolds, using super-resolution microscopy (i.e. STORM).
This work provides original biophysical recipes and leads to new knowledge in the relationship between biophysical cues and chromatin organization, providing insights into how microscale environmental changes affect cell behavior.