Visualization of Fibrillar Collagen Networks Using Label-Free Nonlinear Optical Microscopy: Deciphering the ECM Structure of 3D-Bioprinted Constructs

Abstract

Biological tissues are not exclusively composed of cells. A substantial part of their volume is extracellular space, largely filled by an intricate network of macromolecules. Collagens are one of the main components of this complex network, called extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the basic mechanisms involved in the ECM remodelling of tissues provides a window to advance the development of in vitro tissue models and enhance tissue engineering applications. In this study, we leveraged one label-free nonlinear optical microscopy imaging modality with a novel microfluidic-based three-dimensional (3D) bio-printing technology to investigate the fundamental effects of in vitro model design on the cellular ability to remodel fibrillar collagen. Label-free second harmonic generation (SHG) microscopy allowed us to track fibrillar collagen deposition, and our preliminary data indicated that specific geometries allow cells to remodel ECM more efficiently. Additionally, SHG presents the potential to assess the ECM network development over time, providing important quantitative information crucial to inform artificial tissue development strategies further. Being able to correlate the ECM deposition with cellular behaviour, migration, and even responses to treatments, opens the door to enable the biofabrication of complex networks able to mimic a variety of tissue types and pathological conditions.