Electrochemical evidence for asialoglycoprotein receptor – mediated hepatocyte adhesion and proliferation in three dimensional tissue engineering scaffolds

Abstract

Asialoglycoprotein receptor (ASGPR) is one of the recognition motifs on the surface of hepatocytes, which promote their adhesion to extracellular matrix in liver tissue and appropriate artificial surfaces. ASGPR-mediated adhesion is expected to minimize trans-differentiation of hepatocytes in vitro that is generally observed in integrin-mediated adhesion. The aim of the present study is to verify the role of ASGPR in hepatocyte adhesion and proliferation in scaffolds for hepatic tissue engineering. Scanning Electrochemical Microscopy (SECM) is emerging as a suitable non-invasive analytical tool due to its high sensitivity and capability to correlate the morphology and activity of live cells. HepG2 cells and rat primary hepatocytes cultured in Polyvinyl alcohol (PVA)/Gelatin hydrogel scaffolds with and without galactose (a ligand for ASGPR) modification are studied using SECM. Systematic investigation of live cells cultured for different durations in scaffolds of different compositions (9:1 and 8:2 PVA:Gelatin with and without galactose) reveals significant improvement in cell–cell communication and proliferation on galactose incorporated scaffolds, thereby demonstrating the positive influence of ASGPR-mediated adhesion. In this work, we have also developed a methodology to quantify the respiratory activity and intracellular redox activity of live cells cultured in porous tissue engineering scaffolds. Using this methodology, SECM results are compared with routine cell culture assays viz., MTS ((1-Oxyl-2,2,5,5,-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate) and Albumin assays to demonstrate the better sensitivity of SECM. In addition, the present study demonstrates SECM as a reliable and sensitive tool to monitor the activity of live cells cultured in scaffolds for tissue engineering, which could be used on a routine basis.