(671c) Transitional Liver Models for the Investigation of Chemical and Mechanical Cues on the Progression of Fibrosis

Liver fibrosis often results in hepatocellular carcinomas, multiple organ failure, coma, and is the tenth leading cause of death in the United States. Alcohol abuse, obesity, diabetes and hepatitis C viral infections are some initiating events that induce fibrosis. Fibrosis occurs when uncontrolled wound healing results in the excessive accumulation of extracellular matrix (ECM) components, resulting in scar tissue. The principal liver cell type, the hepatocytes, performs a majority of the organâ??s metabolic and biotransformation functions. However, the nonparenchymal cells (NPCs) are primarily responsible for the initiation and progression of fibrosis. These include the liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs) and hepatic stellate cells (HSCs).

The Space of Disse is a protein rich interface that separates the NPCs from the hepatocytes. This membrane is primarily composed of ECM components and increases 6-10 fold in stiffness as a result of fibrosis. There is little information on how changes in the material microenvironment result in the progression of the disease. Since liver fibrosis occurs over time, we seek to investigate how the physical and chemical properties of the microenvironment gradually transition a healthy tissue into a diseased organ. Our focus is to design transitional liver models wherein the biomaterial properties are presented to cells as a gradient, thereby sending cues to gradually change the cellular profiles from healthy to fibrotic.

We have designed a biopolymeric membrane comprised of ECM components to act as a Space of Disse mimic. This membrane is assembled from alternating layers of collagen (COL; type 1) and hyaluronic acid (HA). The hydrated thickness of this membrane is approximately 700 nm, 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 COL/HA membranes whose elastic moduli ranged from approximately 21 kPa to 43 kPa.

Changes to the ECM composition also occur during hepatic fibrosis. Laminin, an ECM protein, increases in concentration in fibrotic livers. Therefore, we modified the COL/HA membranes with rhodamine-conjugated laminin. NPCs were seeded on unmodified and laminin-conjugated COL/HA membranes and monitored for changes in proliferation, cytokine secretion, migration, and morphology. The effect of membrane stiffness on hepatocytes was investigated by overlaying a monolayer of cells with COL/HA films of varying stiffness.

LSECs and KCs monocultures were seeded on laminin-conjugated or unmodified COL/HA membranes. LSECs exhibited an approximately 36% increase in proliferation on laminin-conjugated PEMs, whereas KC proliferation was approximately 66%. No significant proliferation of either cell type occurred on the unmodified membranes. These results clearly demonstrate that laminin induced proliferation of NPCs; especially the inflammatory KCs. Hepatocytes overlaid with stiffer PEMs exhibited cell death and rounded morphology. The relative viability of hepatocytes decreased by 29%. Combined, these results demonstrate the importance of the chemical and mechanical properties of the microenvironment in the development of liver fibrosis. Ongoing efforts are focused on designing membranes that exhibit chemical and mechanical gradients as well as conjugating additional proteins such as high mobility group box proteins (HMGB1). We are investigating how NPCs, specifically, HSCs are affected by HMGB1, a protein found in fibrotic hepatic tissues.