Mechanisms of hepatocyte attachment to keratin biomaterials Jillian R. Richter, Roche C. de Guzman, Mark E. Van Dyke * Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA article info Article history: Received 6 May 2011 Accepted 25 June 2011 Available online xxx Keywords: Keratin Hepatocyte Cell adhesion Integrin Glycoprotein receptor Focal adhesion abstract Keratin biomaterials support cellular adhesion, proliferation and migration, which have led to their exploitation in a variety of biomedical applications. The mechanism of cell adhesion to keratin bioma- terials, however, is poorly understood. Therefore, the goal of this work was to investigate the mecha- nisms by which human hair keratin-based biomaterials facilitate cellular adhesion. Hepatocytes were used as a model cell type due to the abundance of published data on cell adhesion mechanisms and their relatively copious attachment to keratin substrates. The roles of b 1 - and b 2 -integrins and the hepatic asialoglycoprotein receptor (ASGPR) in hepatocyte adhesion to keratin substrates were studied using attachment assays with and without function blocking antibodies. Blocking of the hepatic integrin subunits did not decrease hepatocyte attachment to keratin. Furthermore, adhesion to keratin did not result in the formation of focal complexes or focal adhesions, nor did it produce an upregulation of phosphorylated-focal adhesion kinase. However, inhibition of hepatic ASGPR decreased the ability of hepatocytes to attach to keratin substrates, which is indicative of the role of this glycoprotein receptor in hepatocyte binding to keratin biomaterials. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Cellular interactions with the extracellular matrix (ECM) play a critical role in cell survival and function. These interactions are facilitated through specific recognition of ECM binding domains by cell surface receptors and lead to a cascade of signaling events that act to mediate cell adhesion [1], migration [2], proliferation [3], differentiation [4] and apoptosis [5]. Recently, the under- standing of such biological mechanisms has become of great interest for biomaterials researchers aiming to control cellular behavior by artificially recapitulating natural cellematrix interactions. Biomaterials derived from human hair keratins have previously been shown to support cellular attachment [6], proliferation [7e10] and migration [11], suggesting that regenerated keratin substrates may mimic the physical and biochemical roles of native ECM. Keratins contain peptide binding motifs such as leucine-aspartic acid-valine (LDV) that have been suggested by other researchers to facilitate cellular adhesion [7,8]. However, the specific mecha- nisms responsible for cellular adhesion and subsequent bioactivity have not been thoroughly investigated. In this work, primary rat hepatocytes were used as a model cell type to study the mecha- nisms by which keratins promote cellular adhesion. Hepatocytes contain a variety of integrin and non-integrin adhesion molecules that aid in their binding to ECM [12]. In normal hepatic lobules, hepatocytes are in direct contact with the perisinusoidal space which contains an abundant amount of fibronectin and small discrete bundles of type I and type IV collagens. Laminin is also present in the hepatic basement membranes and surrounds the central vein of the heptic lobule [13]. Hepatocytes readily interact with the arginine-glycine- aspartic acid (RGD) binding domains of these matrix components via integrin receptors, most notably the a v b 1 and a 1 b 1 integrins. In addition, rat hepatocytes express a 2 b 1 , a 3 b 1 , a 5 b 1 , b 2 -integrins and ICAMs although their involvement in attachment and spreading on ECM molecules has been shown to be less important [14,15]. Hepatocytes also contain non-integrin receptors such as hep- aran sulfate proteoglycans (i.e. syndecans) that recognize matrix fibronectin and laminin via their heparan binding domains and serve as important co-receptors for facilitating hepatocyte inter- actions with these matrix proteins [12,16]. Lectins are also present on the surface of hepatocytes and are responsible for mediating their adhesion to glycoproteins bearing glycan chains that lack sialic acid, termed asialoglycoproteins. Physiologically, the hepatic asialoglycoprotein receptor (ASGPR; also known as the Ashwell- eMorell receptor) facilitates the capture and removal of a wide range of exogenously administered and potentially deleterious glycoproteins with galactose or N-acetylgalactosamine residues at their termini. In addition, hepatic ASGPR has been found to mitigate the lethal coagulopathy of sepsis following systemic Streptococcus * Corresponding author. Tel.: þ1 336 713 7266; fax: þ1 336 713 7290. E-mail address: mavandyk@wfubmc.edu (M.E. Van Dyke). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.06.061 Biomaterials xxx (2011) 1e7 Please cite this article in press as: Richter JR, et al., Mechanisms of hepatocyte attachment to keratin biomaterials, Biomaterials (2011), doi:10.1016/j.biomaterials.2011.06.061