82 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2005, The Society for Biotechnology, Japan Vol. 100, No. 1, 82–87. 2005 DOI: 10.1263/jbb.100.82 Cell Adhesion Ability of Artificial Extracellular Matrix Proteins Containing a Long Repetitive Arg-Gly-Asp Sequence Hiroyuki Kurihara 1 and Teruyuki Nagamune 1 * Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 1 Received 31 January 2005/Accepted 25 March 2005 We generated recombinant artificial extracellular matrix (ECM) proteins containing repetitive Arg-Gly-Asp (RGD) sequences: double (RGD2), 21 (RGD21) and 43 (RGD43) repeats of RGD. RGD43-coated glass slides promoted fibroblast NIH3T3 cell adhesion and spreading on the sur- face. Since actin stress fibers and focal contacts were also observed in cells adhering on RGD43- coated glass slides, it was suggested that the RGD peptides in RGD43 transmitted an adhesion signal via integrins and promoted cell adhesion. We coated recombinant ECM proteins, each con- taining repetitive RGD domains, on polystyrene plates and investigated the effects of RGD length on the cell adhesion ability using three different cell lines, namely, fibroblast NIH3T3, HeLa cancer and neuronal PC12 cell lines. The results indicated that RGD43 had a cell adhesion ability superior to those of natural extracellular matrix proteins, fibronectin and laminin, although the effects of RGD repeat length on the cell adhesion ability depended on the cell line. As an artificial three-dimensional scaffold for cell cultivation, we also prepared an RGD43 hydrogel by a cross- linking reaction using glutaraldehyde. On the RGD43 hydrogel scaffold, fibroblast cells also suc- cessfully adhered under serum-free conditions. [Key words: cell adhesion, fibronectin, RGD motif, tissue engineering, protein polymer, overlap elongation PCR] Integrin-mediated adhesion to extracellular matrix (ECM) proteins is required for the survival of many anchorage- dependent cells (1). Anchorage-dependent cells recognize ECM proteins by their integrins, cell surface receptors, the signal of which activates cytoskeletal remodeling and cell cycle progression (2). The disruption of the cell-ECM com- plex may be crucial for the regulation of the survival machinery, resulting in apoptosis (1, 3). Therefore, natural ECM proteins are required for various cell applications such as tissue engineering. However, natural ECM proteins are huge molecules (e.g., fibronectin: 250 kDa); therefore, it is difficult to produce them in a full-length form using a mi- crobial expression system. The differences in codon usage between mammals and bacteria are also a barrier to produc- ing them using this system. To overcome these problems, artificial ECM-like materials with integrin ligand sequences have been developed as a substitute for natural ECM pro- teins because integrin recognizes a short peptide sequence itself (4, 5). As a protein-based material, an artificial ECM protein, in which a multiple Arg-Gly-Asp (RGD) integrin ligand was introduced between silk-fibroin motifs, Gly-Ala- Gly-Ala-Gly-Ser, has been reported (6). Synthetic polymer- based material with an RGD cell-adhesive-peptide sequence have also been reported (7, 8). These artificial materials can be used as plate modifiers due to their strong cell adhesive ability. Previously, we generated recombinant artificial ECM pro- teins containing RGD repeats of various lengths: 2, 21 and 43 repeat RGD sequences, designated as RGD2, RGD21 and RDG43, respectively (9). It was also shown that RGD43 formed a self-assembled fibrous structure and had an excel- lent cell adhesive ability for NIH3T3 cells. In this study, we quantitatively investigated the effects of RGD repeat length on the cell adhesive ability using three cell lines, namely, fibroblast NIH3T3, HeLa cancer and neuronal PC12 cell lines. We also prepared an RGD43 hydrogel by a chemical cross-linking method, and determined whether this hydrogel can be used as an artificial three-dimensional ECM-like scaffold. MATERIALS AND METHODS Preparation of expression plasmids The expression plas- mids for RGD2, RGD21 and RGD43 were prepared as reported (9). We prepared a novel recombinant artificial ECM protein con- taining Ile-Gly-Asp (IGD) repetitive sequences as a negative con- trol. It was reported that the IGD peptide sequence was first identi- fied from fibronectin and exhibited cell migration, but not cell adhesion (10). To generate DNAs encoding a repetitive IGD pep- tide sequence, we used overlap elongation PCR (9). Briefly, we synthesized DNA oligonucleotides encoding double repeats of the IGD sequence: 5′ ATCGGTGATATCGGTGAT (sense strand) and 5′ ATCACCGATATCACCGAT (antisense strand). The ther- mal cyclic reaction was performed for 40 cycles with DNA oligo- nucleotides, dNTP and DNA polymerase (Vent DNA polymerase; New England Biolabs, Beverly, MA, USA). The resulting elon- * Corresponding author. e-mail: nagamune@bio.t.u-tokyo.ac.jp phone: +81-(0)3-5841-7356 fax: +81-(0)3-5841-8657