Induction of Hepatocyte Differentiation by the Extracellular Matrix and an RGD-Containing Synthetic Peptide
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DIFFERENTIATION BY THE EXTRACELLULAR INDUCTION OF HEPATCY MATRIX AND AN RGD-CONTAINING SYNTHETIC PEPTIDE
David J. Mooney and Robert Langer, Dept. Chemical Engineering, MIT, 77 Massachusetts Ave., Cambridge, MA 02139 Linda K. Hansen, Joseph P. Vacanti, and Donald E. Ingber, Dept. of Surgery, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 ABSTRACT To design novel biomaterials for hepatocyte transplantation it will be necessary to determine whether specific extracellular matrix (ECM) molecule(s) or the adhesive interactions between the surface and hepatocytes are responsible for regulation of hepatocyte function. Purified ECM molecules (laminin, fibronectin, types I and IV collagen) and a synthetic peptide containing the arginine-glycine-aspartate (RGD) cell-binding sequence were precoated at defined densities to non-adhesive polystyrene dishes. Hepatocytes cultured on dishes coated with a low density of ECM molecules (1 nglcm 2 ) maintained a round morphology, and high liver-specific protein secretion rates. In contrast, culturing hepatocytes on increasing ECM densities (50-1000 ng/cm 2 ) resulted in extensive cell spreading, a loss of liver-specific protein secretion, and cell growth. Hepatocytes cultured on dishes coated with the RGD-containing peptide did not spread even on a high density of the peptide (10,000 ng/cm 2 ), and albumin secretion remained high for hepatocytes cultured on all peptide densities (1-10,000 ng/cm 2 ). These results suggest that a variety of ECM molecules and synthetic peptides are capable of inducing hepatocyte differentiation in vitro, and these effects depend on their ability to promote cell spreading. INTRODUCI'ON One proposed therapy to replace lost or deficient tissue function is to engineer new tissue by transplanting selected cell populations attached to a polymer scaffold [1]. However, to induce the proper function in the transplanted cells it will be necessary to understand how the attachment matrix controls cell function, and to design the biomaterial appropriately. Hepatocytes are an ideal cell type to study this question as there is a great deal of interest in utilizing them in tissue engineering [2], and both their growth and expression of liver-specific functions are very responsive to the attachment surface [3]. Hepatocyte viability and function in vitro appear to largely depend on the adhesive interactions between the cells and naturally occurring extracellular matrix (ECM) molecules [3]. However, these molecules present difficulties for large-scale use. They must be isolated from human or animal tissue, and thus are quite expensive and suffer from large batch to batch variations. In addition, they are often not amenable to processing, and typically have poor structural properties. If the mechanisms by which ECM molecules control cell function can be determined, it may be possible to design synthetic analogues to these molecules. These synthetic analogues could be coated onto polymer scaffolds, or covalently bound to polymer scaffolds (i.e., utilizing reactive s
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