(19d) Investigating the Mechanical Microenvironment on Fibrogenesis in Multi-Cellular Hepatic Models

Authors: 
Orbach, S., Virginia Tech
Ford, A., Virginia Tech
Saverot, S. E., Virginia Polytechnic Institute and State University
Rajagopalan, P., Virginia Tech
Chronic liver fibrosis often results in hepatocellular carcinoma or complete organ dysfunction and is the twelfth leading cause of death in the United States. The disease is induced through such conditions as alcohol abuse, obesity, diabetes and hepatitis C viral infections. Fibrosis occurs when uncontrolled wound healing causes excessive accumulation of extracellular matrix (ECM) components, forming scar tissue. In the liver, the nonparenchymal cells (NPCs), such as hepatic stellate cells (HSCs) and Kupffer cells (KCs), are primarily responsible for the initiation and progression of fibrosis. Once HSCs become activated they secrete ECM proteins that increase the stiffness of the tissue. Hardening of liver tissue subsequently affects the functions of hepatocytes, the principal hepatic cell type as well as the liver sinusoidal endothelial cells (LSECs).

Currently, there are very few multi-cellular in vitro models that can be used to investigate hepatic fibrosis. There is little information on how changes in the material microenvironment result in the progression of the disease. We investigated how the physical properties of the microenvironment gradually change a tissue from healthy to fibrotic. We designed multi-cellular liver models using biopolymeric membranes that mimic the Space of Disse, the protein rich interface that separates the hepatocytes from the NPCs. The biopolymeric membrane was assembled with alternating layers of type I collagen and hyaluronic acid. The membranes were designed with a gradient in elastic moduli to emulate the varying stiffness profile in a fibrogenic liver. The hydrated thickness of this membrane was approximately 1.1 μm, which is similar to the height of the Space of Disse in vivo. The membranes were cross-linked in order to vary the rigidity of the membrane to mimic healthy and fibrotic liver tissues. Increasing the cross-linking time resulted in membranes with a gradient of elastic moduli ranging from approximately 21 kPa to 43 kPa.

Multi-cellular liver models were assembled with the gradient membranes.The ratios of hepatocytes to NPCs were analyzed, as NPCs are proliferative during fibrogenesis. After 8 days in culture, at 21 kPa, the hepatocyte to LSEC (~6:1) and hepatocyte to HSC (~16:1) ratios were similar to healthy livers in vivo. The transition of HSCs from a quiescent to an activated state was identified through a dual immunostain for glial fibrillar acidic protein (GFAP) and alpha smooth muscle actin (α-SMA). The effect of substrate stiffness on apoptosis, changes in the secretion of pro-fibrogenic (TGF-β), expression of CD31 in LSECs, and secretion of ECM proteins were also investigated.

Our data demonstrate that a gradual increase in the elastic moduli of the tissue microenvironment can lead to a significant increase in markers of hepatic fibrosis. These models can serve as a platform for future investigations into liver fibrosis.