Fabrication of soft biological materials for engineering multi-scale microphysiological system

Abstract

The conventional use of animal testing models in drug development and physiological research has limitations due to significant species differences, ethical concerns, and poor screening efficiency. Recognizing these limitations, there is a widespread acknowledgment of the imperative need to develop alternative methods that can replace, reduce, and refine the use of animal models. Among the potential alternatives, microphysiological systems (MPS) have emerged as a prominent solution, showcasing remarkable capabilities in faithfully emulating human physiology within biological microenvironments. In the fabrication of MPS, the implementation of mechanical cues of in vivo microenvironments is particularly crucial for accurately replicating physiological phenomena such as mechano-transduction, tissue motility, and morphomechanics. Specifically, the mechanical characteristics of the extracellular matrix (ECM), such as stiffness and stretchability, are known to play a pivotal role in governing mechanobiological behaviors. In this context, soft biological materials, especially ECM hydrogels derived from animal tissues, emerge as promising candidates for MPS fabrication due to their ability to replicate mechanical and biochemical characteristics akin to in vivo ECM. However, current fabrication technologies of ECM hydrogel, including 3D bioprinting, face challenges in achieving micro- to meso-scale features with ECM hydrogels due to its softness. Especially, the limited fabrication technologies of ECM hydrogels pose a challenge in reconstructing unique structures of tissue interfaces (e.g., blood-brain barrier interface, intestinal epithelial interface, glomerular interface etc.), characterized by thin structure in micro-scale or folded structure in meso-scale. Considering the significant roles of the interfacial tissues in human physiology, physiological tissue interface models, especially in the view of mechanobiology, are desired to be reconstructed on in vitro system by using the functional ECM hydrogel. Motivated by difficulties in reconstructing tissue interface models with ECM hydrogel due to the limited fabrication methods, we developed innovative fabrication strategies of ECM hydrogels for the mechanobiological reconstruction of tissue interfaces. In chapter II, we developed an ECM membrane mimicking the in vivo basement membrane (BM), named as NaRE (Nanofiber-reinforced ECM hydrogel) membrane, for the mechanobiological reconstruction of the micro-thin tissue interface model (thickness in the order of 1 μm). The NaRE membrane, reinforced with an electrospun nanofiber scaffold, exhibits mechanical properties similar to the in vivo BM, with a stiffness in the order of 100 kPa and notable stretchability. Strengths of the NaRE membrane for mechanobiological reconstruction of micro-thin tissue interface models were demonstrated by developing a blood-brain barrier (BBB) model on the NaRE membrane, showcasing physiological mechano-transduction of brain microvascular endothelial cells resulting in superior barrier functions. Additionally, we could emulate peristalsis-like dynamic tissue motility on a colon epithelial interface model thanks to the outstanding stretchability of the NaRE membrane. In chapter III, we developed an innovative approach for the mechanobiological reconstruction of a folded tissue interface in meso-scale using an ECM hydrogel. The approach involves the use of an epithelial bilayer composed of epithelium and ECM hydrogel and compression-induced bilayer wrinkling theory. We successfully reconstructed repetitive and regular wrinkles or a deep single fold of epithelial bilayer having a length scale in the order of 100 μm. Through comprehensive experiments and theoretical modeling, we identified that the folding is derived from an intricate interplay between the mechanical characteristics of the epithelium and the ECM hydrogel, specifically strain-stiffening and poroelasticity. It is finally noted that this is the first emulation of the mechanobiological folding of epithelium and ECM on in vitro system. In conclusion, the study offers novel fabrication techniques for thin or folded structures of tissue interfaces using ECM hydrogels, enabling the reconstruction of multi-scale tissue interface models with mechanobiological phenomena.